Methods of diagnosing and treating pre-eclampsia or eclampsia

ABSTRACT

Disclosed herein are methods for diagnosing pre-eclampsia and eclampsia or a propensity to develop pre-eclampsia or eclampsia by detecting the levels of placental growth factor in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/771,518, filed Feb. 4, 2004, which is a continuation-in-partof U.S. patent application Ser. No. 10/624,809, filed Jul. 21, 2003,which claims the benefit of U.S. Provisional Application Nos. 60/467,390filed on May 2, 2003, 60/451,796, filed on Mar. 3, 2003, and 60/397,481,filed on Jul. 19, 2002, each of which is herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

In general, this invention relates to the detection and treatment ofsubjects having pre-eclampsia or eclampsia.

BACKGROUND OF THE INVENTION

Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria thataffects 5 to 10% of pregnancies and results in substantial maternal andfetal morbidity and mortality. Pre-eclampsia accounts for at least200,000 maternal deaths worldwide per year. The symptoms ofpre-eclampsia typically appear after the 20^(th) week of pregnancy andare usually detected by routine ing of the woman's blood pressure andurine. However, these monitoring methods are ineffective for diagnosisof the syndrome at an early stage, which could reduce the risk to thesubject or developing fetus, if an effective treatment were available.

Currently there are no known cures for pre-eclampsia. Pre-eclampsia canvary in severity from mild to life threatening. A mild form ofpre-eclampsia can be treated with bed rest and frequent monitoring. Formoderate to severe cases, hospitalization is recommended and bloodpressure medication or anticonvulsant medications to prevent seizuresare prescribed. If the condition becomes life threatening to the motheror the baby the pregnancy is terminated and the baby is deliveredpre-term.

The proper development of the fetus and the placenta is mediated byseveral growth factors. One of these growth factors is vascularendothelial growth factor (VEGF). VEGF is an endothelial cell-specificmitogen, an angiogenic inducer, and a mediator of vascular permeability.VEGF has also been shown to be important for glomerular capillaryrepair. VEGF binds as a homodimer to one of two homologousmembrane-spanning tyrosine kinase receptors, the fms-like tyrosinekinase (Flt-1) and the kinase domain receptor (KDR), which aredifferentially expressed in endothelial cells obtained from manydifferent tissues. Flt-1, but not KDR, is highly expressed bytrophoblast cells which contribute to placental formation. Placentalgrowth factor (PlGF) is a VEGF family member that is also involved inplacental development. PlGF is expressed by cytotrophoblasts andsyncytiotrophoblasts and is capable of inducing proliferation,migration, and activation of endothelial cells. PlGF binds as ahomodimer to the Flt-1 receptor, but not the KDR receptor. Both PlGF andVEGF contribute to the mitogenic activity and angiogenesis that arecritical for the developing placenta.

A soluble form of the Flt-1 receptor (sFlt-1) was recently identified ina cultured medium of human umbilical vein endothelial cells and in vivoexpression was subsequently demonstrated in placental tissue. sFlt-1 isa splice variant of the Flt-1 receptor which lacks the transmembrane andcytoplasmic domains. sFlt-1 binds to VEGF with a high affinity but doesnot stimulate mitogenesis of endothelial cells. sFlt-1 is believed toact as a “physiologic sink” to down-regulate VEGF signaling pathways.Regulation of sFlt-1 levels therefore works to modulate VEGF and VEGFsignaling pathways. Careful regulation of VEGF and PlGF signalingpathways is critical for maintaining appropriate proliferation,migration, and angiogenesis by trophoblast cells in the developingplacenta. There is a need for methods of accurately diagnosing subjectsat risk for or having pre-eclampsia, particularly before the onset ofthe most severe symptoms. A treatment is also needed.

SUMMARY OF THE INVENTION

We have discovered a means for diagnosing and effectively treatingpre-eclampsia and eclampsia.

Using gene expression analysis, we have discovered that levels of sFlt-1are markedly elevated in placental tissue samples from pregnant womensuffering from pre-eclampsia. sFlt-1 is known to antagonize VEGF andPlGF by acting as a “physiologic sink” and, in pre-eclamptic oreclamptic women, sFlt-1 may be depleting the placenta of necessaryamounts of these essential angiogenic and mitogenic factors. ExcesssFlt-1 may also lead to eclampsia by disrupting the endothelial cellsthat maintain the blood-brain barrier and/or endothelial cells liningthe choroids plexus of the brain thus leading to cerebral edema and theseizures seen in eclampsia. In the present invention, compounds thatincrease VEGF and PlGF levels are administered to a subject to treat orprevent pre-eclampsia or eclampsia by countering the effects of elevatedsFlt-1. In addition, antibodies directed to sFlt-1 are used tocompetitively inhibit binding of VEGF or PlGF to sFlt-1, therebyincreasing the levels of free VEGF and PlGF. RNA interference andantisense nucleobase oligomers are also used to decrease the levels ofsFlt-1. The present invention provides for the use and monitoring ofsFlt-1, VEGF, and PlGF as detection tools for early diagnosis andmanagement of pre-eclampsia or eclampsia, or a predisposition thereto,or a cardiovascular condition, or a predisposition thereto.

We have also discovered that PlGF levels in the urine can be used as adiagnostic tool to detect pre-eclampsia or eclampsia, or apredisposition thereto. The free form of PlGF has an average molecularweight of about 30 kDa and is small enough to be filtered by the kidneyand released into the urine. PlGF, when complexed to sFlt-1, has a muchgreater molecular weight and would therefore not be released into theurine. When the levels of sFlt-1 are increased, sFlt-1 can complex toPlGF, thereby reducing the levels of free PlGF released into the urine.As a result, urine analysis for free PlGF levels can be used to diagnosepre-eclampsia or eclampsia or a patient at risk for having the same.

Accordingly, in one aspect, the invention features a method ofdiagnosing a subject as having, or having a propensity to develop,pre-eclampsia or eclampsia that includes measuring the level of freePlGF in a urine sample from the subject. This method can be used todetermine absolute levels of free PlGF that are below a threshold leveland are diagnostic of pre-eclampsia or eclampsia or the propensity todevelop pre-eclampsia or eclampsia. The normal urinary concentration ofurinary PlGF is approximately 400-800 pg/ml during mid-pregnancy. Inpreferred embodiments, a level of free PlGF less than 400 pg/ml,preferably less than 300, 200, 100, 50, or 10 pg/ml is diagnostic ofpre-eclampsia or eclampsia or the propensity to develop pre-eclampsia oreclampsia. This method can also be used to determine relative levels offree PlGF as compared to a reference sample where a decrease (e.g., 10%,20%, 25%, 50%, 75%, 90%, or more) in the level of free PlGF as comparedto a normal reference sample is diagnostic of pre-eclampsia or eclampsiaor the propensity to develop pre-eclampsia or eclampsia. In this case,the normal reference sample can be a prior sample taken from the samesubject or a sample taken from a matched subject (e.g., matched forgestational age) that is pregnant but does not have pre-eclampsia oreclampsia or a propensity to develop pre-eclampsia or eclampsia. Inadditional preferred embodiments, the reference sample is a standard,level, or number derived from such a normal reference sample. Thereference standard or level can also be a value derived from a normalsubject that is matched to the sample subject by at least one of thefollowing criteria: gestational age of the fetus, age of the mother,blood pressure prior to pregnancy, blood pressure during pregnancy, BMIof the mother, weight of the fetus, prior diagnosis of pre-eclampsia oreclampsia, and a family history of pre-eclampsia or eclampsia. Inpreferred embodiments, the measuring is done using an immunologicalassay such as an ELISA, preferably a sandwich ELISA, or a fluorescenceimmunoassay.

In preferred embodiments, the method also includes the steps of (a)measuring the level of at least one of sFlt-1, PlGF, and VEGFpolypeptide in a sample from the subject, where the sample is a bodilyfluid selected from the group consisting of urine, blood, amnioticfluid, serum, plasma, or cerebrospinal fluid, and (b) comparing thelevel of at least one of sFlt-1, PlGF, and VEGF from the subject to thelevel of the same polypeptide in a reference sample, where an increasein the level of sFlt-1 or a decrease in the level of VEGF or PlGFpolypeptide from the subject sample compared to the reference sample isa diagnostic indicator of pre-eclampsia or eclampsia, or a propensity todevelop pre-eclampsia or eclampsia. In preferred embodiments, sFlt-1 orsFlt-1 and PlGF are measured in a serum sample from a subject identifiedby a urine PlGF assay as being at risk for developing pre-eclampsia oreclampsia. Desirably, this method further includes calculating therelationship between the levels of at least one of sFlt-1, VEGF, andPlGF from step (a) above using a metric, where an alteration in thesubject sample relative to the metric in the reference sample diagnosespre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia. Preferably, the metric is a PAAI (as described above) and aPAAI value greater than 20 is a diagnostic indicator of pre-eclampsia oreclampsia. In preferred embodiments, the sFlt-1 is free, bound, or totalsFlt-1, and the PlGF and VEGF are free PlGF and free VEGF.

In another aspect, the invention features a method of diagnosing asubject as having or having a propensity to develop pre-eclampsia oreclampsia that includes the following steps:

(a) obtaining a sample of urine from the subject;

(b) contacting the sample with a solid support, where the solid supportincludes an immobilized first PlGF binding agent, for a time sufficientto allow binding of the first PlGF binding agent with free PlGF presentin the sample;

(c) contacting the solid support after step (b) with a preparation of asecond labeled PlGF binding agent, for a time sufficient to allowbinding of the second labeled PlGF binding agent to the free PlGF boundto the first immobilized PlGF binding agent;

(d) observing the binding of the second labeled PlGF binding agent tothe immobilized PlGF binding agent bound to free PlGF at the positionwhere the PlGF binding agent is immobilized; and

(e) comparing the binding observed in step (d) with the binding observedusing a reference sample, where the reference sample is PlGF at a knownconcentration; and further where a decrease in the binding observed instep (d) compared to the binding observed using a reference sample is adiagnostic indicator of pre-eclampsia or eclampsia or a propensity todevelop pre-eclampsia or eclampsia.

In another related aspect, the invention features a method of diagnosinga subject as having or having a propensity to develop pre-eclampsia oreclampsia, that includes the following steps:

(a) obtaining a urine sample from the subject;

(b) contacting the urine sample with a solid support, wherein the solidsupport comprises a dehydrated labeled PlGF binding agent and animmobilized secondary agent that binds the PlGF binding agent, for atime sufficient for the sample to rehydrate the labeled PlGF bindingagent and to allow binding of free PlGF in the sample to the labeledPlGF binding agent, wherein the free PlGF bound to the labeled PlGFbinding agent can move (e.g., by capillary movement) to the immobilizedsecondary agent;

(c) observing the binding of the free PlGF-PlGF binding agent complex tothe immobilized secondary agent by detecting the presence of the labelat the position where the secondary agent is immobilized; and

(d) comparing the binding observed in step (c) with the binding observedusing a reference sample, wherein the reference sample is PlGF at knownconcentrations ranging from 10 pg/ml-1 ng/ml.

In preferred embodiments of the above two aspects, the label is acalorimetric label (e.g., colloidal gold). In additional preferredembodiments, the agent that binds PlGF is an antibody, or purifiedfragment thereof, or a peptide. Desirably, the antibody or purifiedfragment thereof specifically binds free PlGF. The agent that binds aPlGF agent is desirably an anti-immunoglobulin antibody or fragmentthereof, protein A, or protein G. In one embodiment, the referencesample is a PlGF sample at a known normal concentration and a decreasein the free PlGF in the subject sample as compared to the referencesample is diagnostic of pre-eclampsia or eclampsia or a propensity todevelop pre-eclampsia or eclampsia.

In another aspect, the invention features a method of diagnosing asubject as having or having a propensity to develop pre-eclampsia oreclampsia that includes the following steps:

(a) obtaining a urine sample from the subject;

(b) contacting the sample with a solid support having an immobilizedPlGF binding agent that is detectably labeled in a manner such that thelabel can distinguish between the PlGF when it is bound to free PlGF andwhen it is not bound to free PlGF. Preferred labels include fluorescentlabels. The membrane is exposed to a urine sample obtained from thesubject for a time sufficient to allow binding of the PlGF binding agentto free PlGF present in the sample. The labeled PlGF binding agent boundto the free PlGF is then measured. Such an assay can be used todetermine the relative level of PlGF (e.g., as compared to the levelfrom a reference sample or standard or level) or to determine theabsolute concentration of PlGF as described above. Preferred assays forthe measurement of binding include fluorescence immunoassays.

In another aspect the invention features a method of diagnosing asubject as having or having a propensity to develop pre-eclampsia oreclampsia that includes the following steps:

(a) obtaining a urine sample from the subject;

(b) contacting the sample with a solid support, wherein the solidsupport comprises an immobilized first PlGF binding agent, for a timesufficient to allow binding of the first PlGF binding agent with freePlGF present in the sample;

(c) contacting the solid support after step (b) with a preparation of asecond PlGF binding agent coupled to an enzyme, for a time sufficient toallow binding of the second PlGF binding agent to the PlGF bound to thefirst immobilized PlGF binding agent; and

(d) adding a preparation of a substrate for the enzyme of step (c), fora time and in an amount sufficient to allow the enzyme to convert thesubstrate to a detectable substrate;

(e) observing the level of the detectable substrate; and

(f) comparing the level observed in step (e) with the binding observedusing a reference sample, wherein the reference sample is PlGF at aknown concentration, wherein an alteration in the level observed in step(e) as compared to the reference sample is a diagnostic indicator ofpre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.

In one embodiment, the reference sample is a PlGF sample at a knownnormal concentration and a decrease in the free PlGF in the subjectsample as compared to the reference sample is diagnostic ofpre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.

In preferred embodiments of the above method, the substrate is detectedvisually, by spectrophotometry or by chemiluminescence. In additionalpreferred embodiments, the enzyme is horseradish peroxidase,P-galactosidase, or alkaline phosphatase and the substrate is TMB(tetramethylbenzidine), Xgal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), or 1,2 dioxetane. Inadditional preferred embodiments, the reference sample is a samplehaving a normal concentration of purified PlGF and the subject sampleshows a decrease (10%, 25%, 50%, 75%, 90% or more) compared to thereference sample. In preferred embodiments of this method, the PlGFbinding agent is a purified anti-PlGF antibody, or fragment thereof, ora peptide. Desirably, the purified anti-PlGF antibody, or fragmentthereof specifically binds free PlGF.

In preferred embodiments of any of the above diagnostic methods, thesolid support is a membrane that can be supported on a dipstickstructure or a lateral flow format, examples of which are described inU.S. Pat. No. 6,660,534. In additional preferred embodiments, thesubject is a non-pregnant human, a pregnant human, or a post-partumhuman. In other embodiments of the above aspects, the subject is anon-human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or acat). In one embodiment, the subject is a non-pregnant human and themethod is used to diagnose a propensity to develop pre-eclampsia oreclampsia. In another embodiment, the subject is a human (pregnant ornon-pregnant) with a history of pre-eclampsia and the method is used todiagnose a propensity to develop pre-eclampsia or eclampsia in asubsequent pregnancy. Desirably, the measuring of levels is done on twoor more occasions and a change in the levels between measurements is adiagnostic indicator of pre-eclampsia or eclampsia.

In additional preferred embodiments, the methods to detect PlGF levelsin a urine sample from a subject can be combined with any of thediagnostic methods described below used to measure the level of sFlt-1,PlGF, or VEGF nucleic acid or polypeptide.

In another aspect, the invention provides a method of diagnosing asubject as having, or having a propensity to develop, pre-eclampsia oreclampsia, the method involves measuring the level of sFlt-1, VEGF, orPlGF polypeptide in a sample from the subject.

In a related aspect, the invention provides a method of diagnosing asubject as having, or having a propensity to develop, pre-eclampsia oreclampsia, by determining the levels of at least two of sFlt-1, VEGF, orPlGF polypeptide in a sample from a subject and calculating therelationship between the levels of sFlt-1 VEGF, or PlGF using a metric,where an alteration in the subject sample relative to a referencediagnoses pre-eclampsia or eclampsia in a subject. In preferredembodiments, the method also includes determining the body mass index(BMI), the gestational age (GA) of the fetus, or both and including theBMI or GA or both in the metric. In one embodiment, the metric is apre-eclampsia anti-angiogenic index (PAAI): [sFlt-1/VEGF+PlGF], wherethe PAAI is used as an indicator of anti-angiogenic activity. In oneembodiment, a PAAI greater than 10, more preferably greater than 20, isindicative of pre-eclampsia or eclampsia. In another embodiment, thelevels of sFlt-1, VEGF, or PlGF polypeptide is determined by animmunological assay, such as an ELISA.

In various embodiments of the above aspects, the sample is a bodilyfluid, such as serum or urine. In one embodiment, a level of sFlt-1greater than 2 ng/ml is indicative of pre-eclampsia or eclampsia. Inpreferred embodiments of the above aspects, the level of sFlt-1polypeptide measured is the level of free, bound, or total sFlt-1polypeptide. In additional embodiments, the sFlt-1 polypeptide can alsoinclude sFlt-1 fragments, degradation products, or enzymatic cleavageproducts. In other preferred embodiments of the above aspects, the levelof VEGF or PlGF is the level of free VEGF or PlGF.

In another aspect, the invention provides a method of diagnosing asubject as having, or having a propensity to develop, pre-eclampsia oreclampsia that includes measuring the level of sFlt-1, VEGF, or PlGFnucleic acid molecule in a sample from the subject and comparing it to areference sample, where an alteration in the levels diagnosespre-eclampsia or eclampsia in the subject, or diagnoses a propensity todevelop pre-eclampsia or eclampsia.

In another aspect, the invention provides a method of diagnosing asubject as having, or having a propensity to develop, pre-eclampsia oreclampsia. This method involves determining the nucleic acid sequence ofa sFlt-1, VEGF, or PlGF gene in a subject and comparing it to areference sequence, where an alteration in the subject's nucleic acidsequence that changes the level or the biological activity of the geneproduct in the subject diagnoses the subject with pre-eclampsia oreclampsia, or a propensity to develop pre-eclampsia or eclampsia. In oneembodiment, the alteration is a polymorphism in the nucleic acidsequence.

In various embodiments of any of the above aspects, the sample is abodily fluid (e.g., urine, amniotic fluid, serum, plasma, orcerebrospinal fluid) of the subject in which the sFlt-1, VEGF, or PlGFis normally detectable. In additional embodiments, the sample is atissue or a cell. Non-limiting examples include placental tissue orplacental cells, endothelial cells, leukocytes, and monocytes. In otherembodiments of the above aspects, the subject is a non-pregnant human, apregnant human, or a post-partum human. In other embodiments of theabove aspects, the subject is a non-human (e.g., a cow, a horse, asheep, a pig, a goat, a dog, or a cat). In one embodiment, the subjectis a non-pregnant human and the method is used to diagnose a propensityto develop pre-eclampsia or eclampsia. In other embodiments of the aboveaspects, at least one of the levels measured is the level of sFlt-1(free, bound, or total). In additional embodiments, the level of sFlt-1measured includes the level of sFlt-1 degradation products or enzymaticcleavage products. In other embodiments of the above aspects, when thelevel of VEGF is measured then the level of sFlt-1 or PlGF is alsomeasured. In additional embodiments, the BMI or GA or both is alsomeasured. In various embodiments of the above aspects, an increase inthe level of sFlt-1 nucleic acid or polypeptide relative to a referenceis a diagnostic indicator of pre-eclampsia or eclampsia. In otherembodiments of the above aspects, a decrease in the level of free VEGFpolypeptide or VEGF nucleic acid relative to a reference is a diagnosticindicator of pre-eclampsia or eclampsia. In other embodiments of theabove aspects, a decrease in the level of free PlGF polypeptide or PlGFnucleic acid relative to a reference is a diagnostic indicator ofpre-eclampsia or eclampsia.

In additional embodiments of the above aspects, the levels are measuredon two or more occasions and a change in the levels between themeasurements is a diagnostic indicator of pre-eclampsia or eclampsia. Inone preferred embodiment, the level of sFlt-1 increases from the firstmeasurement to the next measurement. In another preferred embodiment,the level of VEGF or PlGF decreases from the first measurement to thenext measurement.

Any of the methods and kits described herein can be used to diagnosepre-eclampsia or eclampsia or to predict a subsequent pre-eclampsia in apreviously pregnant woman or a woman with a history of pre-eclampsia.

Any of the diagnostic methods described herein can also be used tomonitor pre-eclampsia or eclampsia in the subject. In preferredembodiments, the diagnostic methods are used to monitor the subjectduring therapy or to determine effective therapeutic dosages. In oneembodiment, a decrease in the level of sFlt-1 polypeptide or nucleicacid measured during or after administering therapy relative to thevalue before therapy indicates an improvement in the pre-eclampsia oreclampsia. In a preferred embodiment, a level of sFlt-1 polypeptide lessthan 2 ng/ml indicates an improvement in the pre-eclampsia or eclampsia.In another embodiment, a therapeutic compound is administered in a dosesuch that the level of sFlt-1 polypeptide is less than 2 ng/ml. Inanother embodiment, an increase in the level of VEGF or PlGF polypeptideor nucleic acid measured during or after administering therapy relativeto the value before therapy indicates an improvement in thepre-eclampsia or eclampsia. In yet another embodiment, a decrease in thePAAI value of a subject indicates an improvement in the pre-eclampsia oreclampsia. In preferred embodiments, the PAAI is less than 20, morepreferably less than 10. A decrease in the PAAI can also indicate aneffective dosage of a therapeutic compound. In one example, atherapeutic compound is administered in a dose such that the PAAI isless than 20. In another example, a therapeutic compound is administeredin a dose such that the PAAI is less than 10. In preferred embodiments,the measuring of the levels of sFlt-1, PlGF, or VEGF is done on two ormore occasions and a change in the levels between measurements is usedto monitor therapy or to determine therapeutic dosages of a compound.The diagnostic methods described above that include the measurement offree PlGF in a urine sample can be used to monitor the subject duringtherapy or to determine effective therapeutic dosages. For example, aurine test for PlGF levels as described above can be used on a regularbasis (e.g., monthly, weekly, every other day, daily, or hourly) for theduration of the therapy to monitor the subject. In one embodiment, thetherapeutic compound is administered in a dose such that the PlGFconcentration is greater than 200 pg/ml, 300 pg/ml, 400 pg/ml, 500pg/ml, 600 pg/ml, 700 pg/ml or 800 pg/ml. For monitoring assays usingPlGF, the reference sample will be a concentration of PlGF indicative ofpre-eclampsia (less than 400 pg/ml) and an increase in the PlGFconcentration as compared to the reference sample will indicate aneffective dosage of a therapeutic compound.

In a related aspect, the invention provides a kit for the diagnosis ofpre-eclampsia or eclampsia in a subject comprising a component usefulfor detecting a sFlt-1, VEGF, or PlGF polypeptide, or any combinationthereof. In one embodiment, the component is an assay selected from thegroup consisting of an immunological assay, an enzymatic assay, and acalorimetric assay. In other embodiments of the above aspects, the kitdiagnoses a propensity to develop pre-eclampsia or eclampsia in apregnant or a non-pregnant subject. In preferred embodiments of theabove aspects, the kit detects VEGF, sFlt-1, or PlGF. In other preferredembodiments of the above aspects, when the kit detects VEGF then sFlt-1or PlGF is also detected. In additional preferred embodiments, the kitis used to detect VEGF, sFlt-1 and PlGF and to determine the PAAI of thesample.

In another aspect, the invention provides a diagnostic kit for thediagnosis of pre-eclampsia or eclampsia in a subject that includes anucleic acid sequence, or fragment thereof, selected from the groupconsisting of sFlt-1, VEGF, and PlGF nucleic acid molecule, or asequence complementary thereto, or any combination thereof. In apreferred embodiment, the kit comprises at least two probes for thedetection of an sFlt-1, VEGF, or PlGF nucleic acid molecule.

The invention also provides a kit for the diagnosis of pre-eclampsia oreclampsia in a subject that includes a PlGF binding agent for detectingfree PlGF in a urine sample and instructions for its used for thediagnosis of pre-eclampsia or eclampsia, or a propensity to developpre-eclampsia or eclampsia in a subject. The kit can also include acomponent useful for an assay selected from the following: animmunological assay (e.g., an ELISA) an enzymatic assay or acalorimetric assay. Desirably, the kit includes any of the componentsneeded to perform any of the diagnostic methods described above. Forexample, the kit desirably includes a membrane, where the PlGF bindingagent or the agent that binds the PlGF binding agent is immobilized onthe membrane. The membrane can be supported on a dipstick structurewhere the sample is deposited on the membrane by placing the dipstickstructure into the sample or the membrane can be supported in a lateralflow cassette where the sample is deposited on the membrane through anopening in the cassette.

In preferred embodiments of any of the diagnostic kits described above,the diagnostic kits include a label or instructions for the intended useof the kit components and a reference sample or purified proteins to beused to establish a standard curve. In one embodiment, the diagnostickit is labeled or includes instructions for use in the diagnosis ofpre-eclampsia or eclampsia, or a propensity to develop pre-eclampsia oreclampsia in a subject. In another embodiment, the diagnostic kit islabeled or includes instructions for use in the diagnosis of acardiovascular condition or a propensity to develop a cardiovascularcondition. In yet another embodiment, the diagnostic kit is labeled orincludes instructions for use in therapeutic monitoring or therapeuticdosage determination. In a preferred embodiment, the diagnostic kitincludes a label or instructions for the use of the kit to determine thePAAI of the subject sample and to compare the PAAI to a reference samplevalue. It will be understood that the reference sample values willdepend on the intended use of the kit. For example, the sample can becompared to a normal PAAI reference value or a normal PlGF value,wherein an increase in the PAAI or a decrease in the PlGF value isindicative of pre-eclampsia or eclampsia, or a propensity to developpre-eclampsia or eclampsia. In another example, a kit used fortherapeutic monitoring can have a reference PAAI value or PlGF valuethat is indicative of pre-eclampsia or eclampsia, wherein a decrease inthe PAAI value or an increase in the PlGF value of the subject samplerelative to the reference sample can be used to indicate therapeuticefficacy or effective dosages of therapeutic compounds.

In a related aspect, the invention features a device for diagnosing asubject as having or having a propensity to develop pre-eclampsia oreclampsia that includes a means for comparing the levels of at least oneof sFlt-1, VEGF, and PlGF polypeptides in a sample from a subjectrelative to a reference sample, wherein an alteration in the levels ofat least one of sFlt-1, VEGF, and PlGF diagnoses pre-eclampsia oreclampsia or a propensity to develop pre-eclampsia or eclampsia in thesubject. In a preferred embodiment the device includes a means for usinga metric to compare the levels as at least two of sFlt-1, VEGF, and PlGFpolypeptides.

In a related aspect, the invention features a device for diagnosing asubject as having or having a propensity to develop pre-eclampsia oreclampsia that includes a component for comparing the levels of at leastone of sFlt-1, VEGF, and PlGF nucleic acid molecules in a sample from asubject relative to a reference sample, wherein an alteration in thelevels of at least one of sFlt-1, VEGF, and PlGF diagnoses pre-eclampsiaor eclampsia or a propensity to develop pre-eclampsia or eclampsia inthe subject. In a preferred embodiment the device includes a componentfor using a metric to compare the levels as at least two of sFlt-1,VEGF, and PlGF nucleic acid molecules.

For the purpose of the present invention, the following abbreviationsand terms are defined below.

By “alteration” is meant a change (increase or decrease) in theexpression levels of a gene or polypeptide as detected by standard artknown methods such as those described above. As used herein, analteration includes a 10% change in expression levels, preferably a 25%change, more preferably a 40% change, and most preferably a 50% orgreater change in expression levels. “Alteration” can also indicate achange (increase or decrease) in the biological activity of any of thepolypeptides of the invention (e.g., sFlt-1, VEGF, or PlGF). Examples ofbiological activity for PlGF or VEGF include binding to receptors asmeasured by immunoassays, ligand binding assays or Scatchard plotanalysis, and induction of cell proliferation or migration as measuredby BrdU labeling, cell counting experiments, or quantitative assays forDNA synthesis such as ³H-thymidine incorporation. Examples of biologicalactivity for sFlt-1 include binding to PlGF and VEGF as measured byimmunoassays, ligand binding assays, or Scatchard plot analysis.Additional examples of assays for biological activity for each of thepolypeptides are described herein. As used herein, an alterationincludes a 10% change in biological activity, preferably a 25% change,more preferably a 40% change, and most preferably a 50% or greaterchange in biological activity.

By “antisense nucleobase oligomer” is meant a nucleobase oligomer,regardless of length, that is complementary to the coding strand or mRNAof an sFlt-1 gene. By a “nucleobase oligomer” is meant a compound thatincludes a chain of at least eight nucleobases, preferably at leasttwelve, and most preferably at least sixteen bases, joined together bylinkage groups. Included in this definition are natural and non-naturaloligonucleotides, both modified and unmodified, as well asoligonucleotide mimetics such as Protein Nucleic Acids, locked nucleicacids, and arabinonucleic acids. Numerous nucleobases and linkage groupsmay be employed in the nucleobase oligomers of the invention, includingthose described in U.S. Patent Application Nos. 20030114412 and20030114407, incorporated herein by reference. The nucleobase oligomercan also be targeted to the translational start and stop sites.Preferably the antisense nucleobase oligomer comprises from about 8 to30 nucleotides. The antisense nucleobase oligomer can also contain atleast 40, 60, 85, 120, or more consecutive nucleotides that arecomplementary to sFlt-1 mRNA or DNA, and may be as long as thefull-length mRNA or gene.

By “body mass index” is meant a number, derived by using height andweight measurements, that gives a general indication of whether or notweight falls within a healthy range. The formula generally used todetermine the body mass index is a person's weight in kilograms dividedby a person's height in meters squared or weight (kg)/(height (m))².

By “cardiovascular condition” is meant an event or disorder of thecardiovascular system. Non-limiting examples of cardiovascularconditions include atherosclerosis, primary myocardial infarction,secondary myocardial infarction, angina pectoris (including both stableand unstable angina), congestive heart failure, sudden cardiac death,cerebral infarction, restenosis, syncope, ischemia, reperfusion injury,vascular occlusion, carotid obstructive disease, transient ischemicattack, and the like.

By “compound” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “chimeric antibody” is meant a polypeptide comprising at least theantigen-binding portion of an antibody molecule linked to at least partof another protein (typically an immunoglobulin constant domain).

By “double-stranded RNA (dsRNA)” is meant a ribonucleic acid moleculecomprised of both a sense and an anti-sense strand. dsRNAs are typicallyused to mediate RNA interference.

By “expression” is meant the detection of a gene or polypeptide bystandard art known methods. For example, polypeptide expression is oftendetected by western blotting, DNA expression is often detected bySouthern blotting or polymerase chain reaction (PCR), and RNA expressionis often detected by northern blotting, PCR, or RNAse protection assays.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “gestational age” is meant a reference to the age of the fetus,counting from the first day of the mother's last menstrual periodusually referred to in weeks.

By a “history of pre-eclampsia or eclampsia” is meant a previousdiagnosis of pre-eclampsia or eclampsia or pregnancy inducedhypertension in the subject themselves or in a related family member.

By “homologous” is meant any gene or protein sequence that bears atleast 30% homology, more preferably 40%, 50%, 60%, 70%, 80%, and mostpreferably 90% or more homology to a known gene or protein sequence overthe length of the comparison sequence. A “homologous” protein can alsohave at least one biological activity of the comparison protein. Forpolypeptides, the length of comparison sequences will generally be atleast 16 amino acids, preferably at least 20 amino acids, morepreferably at least 25 amino acids, and most preferably 35 amino acidsor more. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably at least 110 nucleotides. “Homology” can also refer to asubstantial similarity between an epitope used to generate antibodiesand the protein or fragment thereof to which the antibodies aredirected. In this case, homology refers to a similarity sufficient toelicit the production of antibodies that can specifically recognize theprotein at issue.

By “humanized antibody” is meant an immunoglobulin amino acid sequencevariant or fragment thereof that is capable of binding to apredetermined antigen. Ordinarily, the antibody will contain both thelight chain as well as at least the variable domain of a heavy chain.The antibody also may include the CH1, hinge, CH2, CH3, or CH4 regionsof the heavy chain. The humanized antibody comprises a framework region(FR) having substantially the amino acid sequence of a humanimmunoglobulin and a complementarity determining region (CDR) havingsubstantially the amino acid sequence of a non-human immunoglobulin (the“import” sequences).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)₂, Fabc, Fv) in whichall or substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. By“complementarity determining region (CDR)” is meant the threehypervariable sequences in the variable regions within each of theimmunoglobulin light and heavy chains. By “framework region (FR)” ismeant the sequences of amino acids located on either side of the threehypervariable sequences (CDR) of the immunoglobulin light and heavychains.

The FR and CDR regions of the humanized antibody need not correspondprecisely to the parental sequences, e.g., the import CDR or theconsensus FR may be mutagenized by substitution, insertion or deletionof at least one residue so that the CDR or FR residue at that site doesnot correspond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75%,preferably 90%, and most preferably at least 95% of the humanizedantibody residues will correspond to those of the parental FR and CDRsequences.

By “hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences, or portions thereof, undervarious conditions of stringency. (See, e.g., Wahl and Berger (1987)Methods Enzymol. 152:399; Kimmel, Methods Enzymol. 152:507, 1987.) Forexample, stringent salt concentration will ordinarily be less than about750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500mM NaCl and 50 mM trisodium citrate, and most preferably less than about250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridizationcan be obtained in the absence of organic solvent, e.g., formamide,while high stringency hybridization can be obtained in the presence ofat least about 35% formamide, and most preferably at least about 50%formamide. Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. Varying additionalparameters, such as hybridization time, the concentration of detergent,e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion ofcarrier DNA, are well known to those skilled in the art. Various levelsof stringency are accomplished by combining these various conditions asneeded. In a preferred embodiment, hybridization will occur at 30° C. in750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferredembodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mMtrisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmonsperm DNA (ssDNA). In a most preferred embodiment, hybridization willoccur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50%formamide, and 200 μg/ml ssDNA. Useful variations on these conditionswill be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “intrauterine growth retardation (IUGR)” is meant a syndromeresulting in a birth weight which is less that 10 percent of thepredicted fetal weight for the gestational age of the fetus. The currentWorld Health Organization criterion for low birth weight is a weightless than 2,500 gm (5 lbs. 8 oz.) or below the 10^(th) percentile forgestational age according to U.S. tables of birth weight for gestationalage by race, parity, and infant sex (Zhang and Bowes, Obstet. Gynecol.86:200-208, 1995). These low birth weight babies are also referred to as“small for gestational age (SGA)”. Pre-eclampsia is a condition known tobe associated with IUGR or SGA.

By “metric” is meant a measure. A metric may be used, for example, tocompare the levels of a polypeptide or nucleic acid molecule ofinterest. Exemplary metrics include, but are not limited to,mathematical formulas or algorithms, such as ratios. The metric to beused is that which best discriminates between levels of sFlt-1, VEGF, orPlGF in a subject having pre-eclampsia or eclampsia and a normal controlsubject. Depending on the metric that is used, the diagnostic indicatorof eclampsia or pre-eclampsia may be significantly above or below areference value (e.g., from a control subject not having pre-eclampsiaor eclampsia).

sFlt-1 level is measured by measuring the amount of free, bound (i.e.,bound to growth factor), or total sFlt-1 (bound +free). VEGF or PlGFlevels are determined by measuring the amount of free PlGF or free VEGF(i.e., not bound to sFlt-1). One exemplary metric is[sFlt-1/(VEGF+PlGF)], also referred to as the pre-eclampsiaanti-angiogenic index (PAAI).

By “operably linked” is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s).

By “pharmaceutically acceptable carrier” is meant a carrier that isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier substance isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and described,for example, in Remington's Pharmaceutical Sciences, (20^(th) edition),ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.

By “placental growth factor (PlGF)” is meant a mammalian growth factorthat is homologous to the protein defined by GenBank accession numberP49763 and that has PlGF biological activity. PlGF is a glycosylatedhomodimer belonging to the VEGF family and can be found in two distinctisoforms through alternative splicing mechanisms. PlGF is expressed bycyto- and syncytiotrophoblasts in the placenta and PlGF biologicalactivities include induction of proliferation, migration, and activationof endothelial cells, particularly trophoblast cells.

By “polymorphism” is meant a genetic variation, mutation, deletion oraddition in an sFlt-1, PlGF, or VEGF nucleic acid molecule that isindicative of a predisposition to develop the conditions. Suchpolymorphisms are known to the skilled artisan and are described byParry et al. (Eur. J. Immunogenet. 26:321-3, 1999). A polymorphism maybe present in the promoter sequence, an open reading frame, intronicsequence, or untranslated 3′ region of an sFlt-1 gene.

By “pre-eclampsia” is meant the multi-system disorder that ischaracterized by hypertension with proteinuria or edema, or both,glomerular dysfunction, brain edema, liver edema, or coagulationabnormalities due to pregnancy or the influence of a recent pregnancy.Pre-eclampsia generally occurs after the 20^(th) week of gestation.Pre-eclampsia is generally defined as some combination of the followingsymptoms: (1) a systolic blood pressure (BP)>140 mmHg and a diastolicBP>90 mmHg after 20 weeks gestation (generally measured on twooccasions, 4-168 hours apart), (2) new onset proteinuria (1+by dipstikon urinanaysis, >300 mg of protein in a 24-hour urine collection, or asingle random urine sample having a protein/creatinine ratio>0.3), and(3) resolution of hypertension and proteinuria by 12 weeks postpartum.Severe pre-eclampsia is generally defined as (1) a diastolic BP>110 mmHg(generally measured on two occasions, 4-168 hours apart) or (2)proteinuria characterized by a measurement of 3.5 g or more protein in a24-hour urine collection or two random urine specimens with at least 3+protein by dipstick. In pre-eclampsia, hypertension and proteinuriagenerally occur within seven days of each other. In severepre-eclampsia, severe hypertension, severe proteinuria and HELLPsyndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsiacan occur simultaneously or only one symptom at a time. Occasionally,severe pre-eclampsia can lead to the development of seizures. Thissevere form of the syndrome is referred to as “eclampsia.” Eclampsia canalso include dysfunction or damage to several organs or tissues such asthe liver (e.g., hepatocellular damage, periportal necrosis) and thecentral nervous system (e.g., cerebral edema and cerebral hemorrhage).The etiology of the seizures is thought to be secondary to thedevelopment of cerebral edema and focal spasm of small blood vessels inthe kidney.

By “pre-eclampsia anti-angiogenesis index (PAAI)” is meant the ratio ofsFlt-1/JVEGF+PlGF used as an indicator of anti-angiogenic activity. APAAI greater than 10, more preferably greater than 20, is considered tobe indicative of pre-eclampsia or risk of pre-eclampsia.

By “protein” or “polypeptide” or “polypeptide fragment” is meant anychain of more than two amino acids, regardless of post-translationalmodification (e.g., glycosylation or phosphorylation), constituting allor part of a naturally occurring polypeptide or peptide, or constitutinga non-naturally occurring polypeptide or peptide.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of 20% or greater, more preferably of 50% or greater, andmost preferably of 75% or greater, in the level of protein or nucleicacid, detected by the aforementioned assays (see “expression”). Inembodiments that relate to the use of antisense nucleobase oligomers orRNA interference to reduce or inhibit the levels of protein or nucleicacid, the % reduction or inhibition is determined by comparing thelevels in the treated sample to the levels in a sample not treated withantisense nucleobase oligomers or dsRNA.

By “reference sample” is meant a sample taken from a subject prior tothe time of the test sample, a pregnant subject not having pre-eclampsiaor eclampsia, a subject that is pregnant but the sample was taken earlyin pregnancy (e.g., in the first or second trimester or before thedetection of pre-eclampsia or eclampsia), a subject that is pregnant butdoes not have pre-eclampsia or eclampsia and has no history ofpre-eclampsia or eclampsia, or a subject that is not pregnant. Areference sample can also be a purified polypeptide (e.g., PlGF, VEGF,or sFlt-1) at a concentration known to be a normal concentration notdiagnostic of pre-eclampsia or eclampsia. For example, urinary PlGFconcentrations during normal pregnancy may range from 400-800 pg/ml,whereas those with active preeclampsia may be below 200 pg/ml duringmid-gestation. A level of urinary PlGF below 400 pg/ml or below 200pg/ml is indicative of pre-eclampsia or a propensity to developpreeclampsia. A “reference sample” can also be a reference standard orlevel. By “reference standard or level” is meant a value or numberderived from a reference sample. The reference standard or level canalso be a value or number derived from a normal subject that is matchedto the sample subject by at least one of the following criteria:gestational age of the fetus, maternal age, maternal blood pressureprior to pregnancy, maternal blood pressure during pregnancy, BMI of themother, weight of the fetus, prior diagnosis of pre-eclampsia oreclampsia, and a family history of pre-eclampsia or eclampsia. Areference value can also be used which is determined based on the valuesof a particular polypeptide in a reference sample.

By “small interfering RNAs (siRNAs)” is meant an isolated dsRNAmolecule, preferably greater than 10 nucleotides in length, morepreferably greater than 15 nucleotides in length, and most preferablygreater than 19 nucleotides in length that is used to identify thetarget gene or mRNA to be degraded. A range of 19-25 nucleotides is themost preferred size for siRNAs. siRNAs can also include short hairpinRNAs in which both strands of an siRNA duplex are included within asingle RNA molecule. siRNA includes any form of dsRNA (proteolyticallycleaved products of larger dsRNA, partially purified RNA, essentiallypure RNA, synthetic RNA, recombinantly produced RNA) as well as alteredRNA that differs from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. Suchalterations can include the addition of non-nucleotide material, such asto the end(s) of the 21 to 23 nt RNA or internally (at one or morenucleotides of the RNA). In a preferred embodiment, the RNA moleculescontain a 3′hydroxyl group. Nucleotides in the RNA molecules of thepresent invention can also comprise non-standard nucleotides, includingnon-naturally occurring nucleotides or deoxyribonucleotides.Collectively, all such altered RNAs are referred to as analogs of RNA.siRNAs of the present invention need only be sufficiently similar tonatural RNA that it has the ability to mediate RNA interference (RNAi).As used herein, RNAi refers to the ATP-dependent targeted cleavage anddegradation of a specific mRNA molecule through the introduction ofsmall interfering RNAs or dsRNAs into a cell or an organism. As usedherein “mediate RNAi” refers to the ability to distinguish or identifywhich RNAs are to be degraded.

By “soluble Flt-1 (sFlt-1)” (also known as sVEGF-R 1) is meant thesoluble form of the Flt-1 receptor, that is homologous to the proteindefined by GenBank accession number U01134, and that has sFlt-1biological activity. The biological activity of an sFlt-1 polypeptidemay be assayed using any standard method, for example, by assayingsFlt-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and thecytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 canbind to VEGF and PlGF with high affinity, but it cannot induceproliferation or angiogenesis and is therefore functionally differentfrom the Flt-1 and KDR receptors. sFlt-1 was initially purified fromhuman umbilical endothelial cells and later shown to be produced bytrophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1family member or isoform. In additional embodiments, sFlt-1 can alsomean degradation products or fragments that result from enzymaticcleavage of the Flt-1 receptor and that maintain sFlt-1 biologicalactivity. In one example, specific metalloproteinases released from theplacenta may cleave the extracellular domain of Flt-1 receptor torelease the N-terminal portion of Flt-1 into circulation.

By “specifically binds” is meant a compound or antibody which recognizesand binds a polypeptide of the invention but that does not substantiallyrecognize and bind other molecules in a sample, for example, abiological sample, which naturally includes a polypeptide of theinvention. In one example, an antibody that specifically binds sFlt-1does not bind Flt-1.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Included in this definition are pregnant, post-partum, and non-pregnantmammals.

By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (e.g.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein. Preferably, the amino acid sequence is at least 70%,more preferably at least about 80%, and most preferably at least about90% homologous to another amino acid sequence. Methods to determineidentity are available in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux et al., NucleicAcids Research 12:387, 1984), BLASTP, BLASTN, and FASTA (Altschul etal., J. Mol. Biol. 215:403 (1990). The well-known Smith Watermanalgorithm may also be used to determine identity. The BLAST program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 athttp://www.ncbi.nlm.nih.gov/blast/). These software programs matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

By “symptoms of pre-eclampsia” is meant any of the following: (1) asystolic blood pressure (BP)>140 mmHg and a diastolic BP>90 mmHg after20 weeks gestation, (2) new onset proteinuria (1+by dipstik onurinanaysis, >300 mg of protein in a 24 hour urine collection, or randomurine protein/creatinine ratio>0.3), and (3) resolution of hypertensionand proteinuria by 12 weeks postpartum. The symptoms of pre-eclampsiacan also include renal dysfunction and glomerular endotheliosis orhypertrophy. By “symptoms of eclampsia” is meant the development of anyof the following symptoms due to pregnancy or the influence of a recentpregnancy: seizures, coma, thrombocytopenia, liver edema, pulmonaryedema, and cerebral edema.

By “therapeutic amount” is meant an amount that when administered to apatient suffering from pre-eclampsia or eclampsia is sufficient to causea qualitative or quantitative reduction in the symptoms of pre-eclampsiaor eclampsia as described herein. A “therapeutic amount” can also meanan amount that when administered to a patient suffering frompre-eclampsia or eclampsia is sufficient to cause a reduction in theexpression levels of sFlt-1 or an increase in the expression levels ofVEGF or PlGF as measured by the assays described herein.

By “treating” is meant administering a compound or a pharmaceuticalcomposition for prophylactic and/or therapeutic purposes. To “treatdisease” or use for “therapeutic treatment” refers to administeringtreatment to a subject already suffering from a disease to improve thesubject's condition. Preferably, the subject is diagnosed as sufferingfrom pre-eclampsia or eclampsia based on identification of any of thecharacteristic symptoms described below or the use of the diagnosticmethods described herein. To “prevent disease” refers to prophylactictreatment of a subject who is not yet ill, but who is susceptible to, orotherwise at risk of, developing a particular disease. Preferably asubject is determined to be at risk of developing pre-eclampsia oreclampsia using the diagnostic methods described herein. Thus, in theclaims and embodiments, treating is the administration to a mammaleither for therapeutic or prophylactic purposes.

By “trophoblast” is meant the mesectodermal cell layer covering theblastocyst that erodes the uterine mucosa and through which the embryoreceives nourishment from the mother; the cells contribute to theformation of the placenta.

By “vascular endothelial growth factor (VEGF)” is meant a mammaliangrowth factor that is homologous to the growth factor defined in U.S.Pat. Nos. 5,332,671; 5,240,848; 5,194,596; and Charnock-Jones et al.(Biol. Reproduction, 48:1120-1128, 1993), and has VEGF biologicalactivity. VEGF exists as a glycosylated homodimer and includes at leastfour different alternatively spliced isoforms. The biological activityof native VEGF includes the promotion of selective growth of vascularendothelial cells or umbilical vein endothelial cells and induction ofangiogenesis. As used herein, VEGF includes any VEGF family member orisoform (e.g. VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF165,or VEGF 121). Preferably, VEGF is the VEGF121 or VEGF165 isoform(Tischer et al., J. Biol. Chem. 266, 11947-11954, 1991; Neufed et al.Cancer Metastasis 15:153-158, 1996), which is described in U.S. Pat.Nos. 6,447,768; 5,219,739; and 5,194,596, hereby incorporated byreference. Also included are mutant forms of VEGF such as theKDR-selective VEGF and Flt-selective VEGF described in Gille et al. (J.Biol. Chem. 276:3222-3230, 2001). As used herein VEGF also includes anymodified forms of VEGF such as those described in LeCouter et al.(Science 299:890-893, 2003). Although human VEGF is preferred, theinvention is not limited to human forms and can include other animalforms of VEGF (e.g. mouse, rat, dog, or chicken).

By “vector” is meant a DNA molecule, usually derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Arecombinant vector will contain one or more unique restriction sites,and may be capable of autonomous replication in a defined host orvehicle organism such that the cloned sequence is reproducible. A vectorcontains a promoter operably linked to a gene or coding region suchthat, upon transfection into a recipient cell, an RNA is expressed.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an autoradiogram depicting sFlt-1 mRNA and protein expressionlevels in pre-eclampsia. FIG. 1A shows mRNA expression of placentalsFlt-1 from three patients with pre-eclampsia (P1, P2, P3) and threenormotensive term pregnancies (N1, N2, N3) as determined by northernblot analysis. The higher band (7.5 kb) is the full length flt-1 mRNAand the lower, more abundant band (3.4 kb) is the alternatively splicedsFlt-1 mRNA. GAPDH is included as a control and the arrowhead indicates28S RNA. Patients P1 and P2 had severe pre-eclampsia, whereas patient P3had mild pre-eclampsia. FIG. 1B is a graph showing sFlt-1 levels inserum from patients with mild pre-eclampsia (mild PE), patients withsevere pre-eclampsia (severe PE), and normotensive pregnant women atterm (normal). sFlt-1 levels were measured by an ELISA performed forsFlt-1 using a commercially available kit (R & D Systems, Minneapolis,Minn.). Patients with pre-term deliveries for other reasons (pre-term)were included as additional controls to rule out gestational agespecific changes. The number of patients tested is shown in parenthesisin the X axis. Samples were collected prior to delivery (t=0) and 48hours after delivery (t=48). FIG. 1C is a graph showinganti-angiogenesis index ratios (PAAI=sFlt-1/(VEGF+PlGF)) ratios at thetime of delivery (t=0) as determined by ELISA for all the patientsdescribed in FIG. 1B.

FIGS. 2A-2F are photomicrographs showing the anti-angiogenic effect ofexcess sFlt-1 in pre-eclampsia. Endothelial tube assays were performedusing serum from four normal pregnant controls and four patients withpre-eclampsia. A representative experiment from one normal control andone patient with pre-eclampsia is shown. FIGS. 2A, 2B, and 2C showassays performed using serum from a normal patient, while FIGS. 2D, 2E,and 2F show assays performed using serum from a patient withpre-eclampsia. In FIG. 2A, t=0 (10% serum from a normal pregnant womanat term); in FIG. 2B, t=48 (10% serum from normal pregnant woman 48hours after delivery); in FIG. 2C, t=0+exogenous sFlt-1 (10 ng/ml); inFIG. 2D, t=0 (10% serum from pre-eclamptic woman prior to delivery); inFIG. 2E, t=48 (10% serum from pre-eclamptic woman 48 hours afterdelivery); and in FIG. 2F, t=0+exogenous VEGF(10 ng/ml)+PlGF (10 ng/ml).The tube assay was quantitated and the mean tube length +/− SEM is shownin pixels at the bottom of each panel.

FIGS. 3A and 3B are graphs showing that inhibition of VEGF and PlGFinduced vasodilation of renal microvessels by sFlt-1. FIG. 3A shows thatthe increase in relaxation responses of rat renal arterioles to sFlt-1(S), VEGF (V), PlGF (P) was measured at three different doses. V+ and P+represent vasodilatory responses of the individual reagents in thepresence of sFlt-1 at 100 ng/ml. All experiments were done in 6different dissected rat renal microvessels and data is shown as mean +/−SEM. The * represents statistical significance with p<0.01 as comparedto individual reagents alone. FIG. 3B shows the increase in relaxationresponses at physiological doses: VEGF 100 pg/ml (V), PlGF 500 pg/ml(P), sFlt-110 ng/ml (S), VEGF (100 pg/ml)+PlGF 500 pg/ml (V+P) or VEGF(100 pg/ml)+PlGF 500 pg/ml+sFlt-1 10 ng/ml (V+P+S). All experiments weredone in 6 different dissected rat renal microvessels and data is shownas mean +/−SEM. The * represents statistical significance with p<0.05 ascompared with V+P.

FIGS. 4A and 4B are images showing sFlt-1 induction of glomerularendotheliosis. FIG. 4A is photomicrograph showing hematoxylin and eosin(H & E) staining in a capillary occlusion in the sFlt-1 treated animalswith enlarged glomeruli and swollen cytoplasm as compared to controls.“Glomerular endotheliosis” with bubbly cytoplasm is shown in the sFlt-1treated animals on periodic acid schiff (PAS) stain. All lightmicroscopy pictures were taken at 60×, original magnification. FIG. 4Bis an electron micrograph of sFlt-1 treated glomeruli that confirmscytoplasmic swelling of the endocapillary cells. The immunofluorescence(IF) for fibrin pictures were taken at 40× and the EM pictures weretaken at 2400×, original magnification. All figures were reproduced atidentical magnifications.

FIGS. 5A-5C are graphs showing sFlt-1 levels measured before and afterthe onset of pre-eclampsia by gestational age. FIG. 5A is a graphshowing the mean serum concentrations in pg/ml for normotensive controls(lighter line with open triangles), cases before pre-eclampsia (filledcircles), and cases after pre-eclampsia -“endpoint” specimens-(filledsquares) within 4-5 week gestational age windows prior to onset oflabor. Brackets indicate standard error of the mean. Asterisks indicatesignificant differences with respect to control specimens within thesame gestational age window after logarithmic transformation: *p<0.05,**p<0.01, ***p<0.001. FIG. 5B is a graph showing the mean serumconcentrations of sFlt1 in pg/ml for cases before and after the onset ofpre-eclampsia within intervals of weeks before pre-eclampsia. PEindicates the arithmetic mean of 43 endpoint specimens (obtained on orfollowing onset of pre-eclampsia). Mean gestational age (days) isindicated in parentheses below each time interval. The horizontal lineindicates the level in the endpoint specimens. The vertical linesdemarcate the period ≦5 weeks before pre-eclampsia. FIG. 5C is a graphshowing the mean serum concentrations of sFlt-1 in pg/ml by gestationalage windows for normotensive controls and cases before pre-eclampsia,after excluding specimens obtained within 5 weeks of onset ofpre-eclampsia. There are no significant differences.

FIGS. 6A-6C are graphs showing the levels of PlGF before and afterpre-eclampsia by gestational age. FIG. 6A is a graph showing PlGF levelsin all specimens obtained before labor and delivery. Brackets indicatestandard error of the mean. Asterisks indicate significant differenceswith respect to control specimens within the same interval afterlogarithmic transformation: **p<0.01, ***p<0.001. FIG. 6B is a graphshowing the mean serum concentrations of PlGF in pg/ml for cases beforeand after onset of pre-eclampsia within intervals of weeks beforepre-eclampsia. PE indicates the arithmetic mean of 43 endpoint specimens(obtained on or following onset of pre-eclampsia). Mean gestational age(days) is indicated in parentheses below each time interval. Thehorizontal line indicates the level in the endpoint specimens. Thevertical lines demarcate the period ≦5 weeks before pre-eclampsia. FIG.6C is a graph showing the mean serum concentrations of PlGF in pg/ml bygestational age windows for normotensive controls and cases onset ofpre-eclampsia.

FIGS. 7A and 7B are graphs showing sFlt-1 and PlGF levels bypre-eclampsia status and severity. FIG. 7A is a graph showing thearithmetic mean serum concentrations of sFlt-1 (black bars) and PlGF(white bars) at 23-32 weeks of gestation in controls and cases (beforeonset of clinical disease) with mild pre-eclampsia, severepre-eclampsia, pre-eclampsia with onset <37 weeks, pre-eclampsia with asmall for gestational age (SGA) infant, and pre-eclampsia with onset <34weeks. Numbers of specimens are recorded below each column pair.Adjustment for gestational age and body mass index resulted in minorchanges with no affect on level of significance. FIG. 7B is a graphshowing the arithmetic mean serum concentrations of sFlt-1 (black bars)and PlGF (white bars) at 33-41 weeks of gestation in controls and cases(before onset of clinical disease) with mild pre-eclampsia, severepre-eclampsia, pre-eclampsia with onset <37 weeks, and pre-eclampsiawith an SGA infant. Numbers of specimens are recorded below each columnpair. Adjustment for gestational age and body mass index resulted inminor changes.

FIG. 8 is an autoradiogram showing the expression of flt, sFlt-1, andrelated variants or fragments in PBMCs isolated from normal andpreeclamptic patients. Protein lysates were analyzed by western blotsusing an antibody that recognizes the N-terminus of Flt-1 protein.

FIGS. 9A to 9D are graphs showing the concentration of urinary PlGF byintervals of gestational age. FIG. 9A is a graph showing the mean PlGFconcentrations before and after the onset of clinical pre-eclampsiaaccording to gestational age. I bars represent standard errors. FIG. 9Bis a graph showing the mean PlGF expressed as pg per mg creatininebefore and after the onset of clinical pre-eclampsia. I bars representstandard errors. FIG. 9C is a graph showing the mean PlGF concentrationsbefore and after the onset of clinical pre-eclampsia, using only firstmorning urine specimens. I bars represent standard errors. FIG. 9D is agraph showing the mean PlGF concentrations before and after the onset ofclinical pre-eclampsia, using only random urine specimens. I barsrepresent standard errors.

FIG. 10 is a graph showing the mean concentrations of PlGF according topre-eclampsia status and severity, before and after normalization forcreatinine. PlGF concentrations and pg per mg creatinine are shown at21-32 weeks of gestation in controls and in women who later had clinicalpre-eclampsia (PE) according to whether they had mild pre-eclampsia,severe pre-eclampsia, pre-eclampsia with an onset at less than 37 weeksof gestation, pre-eclampsia and a small-for-gestational-age infant(SGA), or pre-eclampsia with an onset at less than 34 weeks ofgestation. Specimens from women in whom pre-eclampsia developed wereobtained before the onset of clinical disease. The P values given arefor the comparisons with the specimens from the controls. I barsrepresent SEs.

FIG. 11 is a graph showing a longitudinal plot of placental growthfactor concentrations within individual women by gestational age.

FIGS. 12A and 12B are graphs showing scatter plots of urinary PlGFconcentrations and ratios of sFlt-1 to PlGF in serum at 21-32 weeks bydays of gestation. Values were obtained from paired urine and serumspecimens obtained from 20 women before development of pre-eclampsia atless than 37 weeks of gestation and from 69 normotensive controls. FIG.12A shows urinary PlGF concentrations. FIG. 12B shows serum ratios ofsFlt1 to PlGF.

FIG. 13 is a graph showing the mean urinary concentrations of placentalgrowth factor (PlGF) in normotensive women with infants not bornsmall-for-gestational-age (SGA), normotensive women with SGA infants,women with gestational hypertension, and women in whom preeclampsiadeveloped before 37 weeks of gestation. Urinary PlGF concentrations inpg/ml and in pg per mg creatinine are shown at 21-32 weeks of gestationin normotensive women whose infants were not born small-for-gestationalage (NT-SGA), normotensive women with SGA infants (NT+SGA), women whosubsequently developed gestational hypertension (GH), and women whosubsequently developed preeclampsia before 37 weeks of gestation (PE<37wks). Specimens from women in whom gestational hypertension orpreeclampsia developed were obtained before the onset of clinicaldisease. The mean gestational age at specimen collection was similar inall groups. N indicates number of specimens. The P values given are forthe comparisons with the specimens from the controls (NT-SGA). I barsrepresent SEs.

DETAILED DESCRIPTION

We have discovered that sFlt-1 levels are elevated in blood serumsamples taken from pre-eclamptic women. sFlt-1 binds to VEGF and PlGFwith high affinity and blocks the mitogenic and angiogenic activity ofthese growth factors. Thus, sFlt-1 is an excellent diagnostic marker forpre-eclampsia and VEGF and PlGF may be used to treat pre-eclampsia.Furthermore, we have discovered therapeutic agents that interfere withsFlt-1 binding to purified VEGF or PlGF, or agents that increase levelsof biologically active VEGF or PlGF, can be used to treat or preventpre-eclampsia or eclampsia in a subject. Such agents include, but arenot limited to, antibodies to sFlt-1, oligonucleotides for antisense orRNAi that reduce levels of sFlt-1, compounds that increase the levels ofVEGF or PlGF, and small molecules that bind sFlt-1 and block the growthfactor binding site. The invention also features methods for measuringlevels of growth factors; the methods can be used as diagnostic toolsfor early detection of pre-eclampsia or an increased risk of developingpre-eclampsia or eclampsia.

While the detailed description presented herein refers specifically tosFlt-1, VEGF, or PlGF, it will be clear to one skilled in the art thatthe detailed description can also apply to sFlt-1, VEGF, or PlGF familymembers, isoforms, and/or variants, and to growth factors shown to bindsFlt-1. The following examples are for the purposes of illustrating theinvention, and should not be construed as limiting.

EXAMPLE 1 Increased Levels of sFlt-1 mRNA and Protein in Pregnant WomenWith Pre-Eclampsia

In an attempt to identify novel secreted factors playing a pathologicrole in pre-eclampsia, we performed gene expression profiling ofplacental tissue from women with and without pre-eclampsia usingAffymetrix U95A microarray chips. We found that the gene for sFlt-1 wasupregulated in women with pre-eclampsia.

In order to confirm the upregulation of sFlt-1 in pre-eclampsia, weperformed Northern blots to analyze the placental sFlt-1 mRNA levels(FIG. 1A) and ELISA assays to measure serum protein levels of sFlt-1(FIG. 1B) in pre-eclamptic pregnant women as compared with normotensivepregnant women. Pre-eclampsia was defined as (1) a systolic bloodpressure (BP)>140 mmHg and a diastolic BP>90 mmHg after 20 weeksgestation, (2) new onset proteinuria (1+by dipstik on urinanalysis, >300mg of protein in a 24 hour urine collection, or random urineprotein/creatinine ratio >0.3, and (3) resolution of hypertension andproteinuria by 12 weeks postpartum. Patients with underlyinghypertension, proteinuria, or renal disease were excluded. Patients weredivided into mild and severe pre-eclampsia based on the presence orabsence of nephritic range proteinuria (>3 g of protein on a 24 hoururine collection or urine protein/creatinine ratio greater than 3.0).The mean urine protein/creatinine ratios in the mild pre-eclampsia groupwere 0.94+/−0.2 and in the severe pre-eclampsia group were 7.8 +/−2.1.The mean gestational ages of the various groups were as follows: normal38.8+/−0.2 weeks, mild pre-eclampsia 34+/−1.2 weeks, severepre-eclampsia 31.3 +/−0.6 weeks, and pre-term 29.5+/−2.0 weeks.Placental samples were obtained immediately after delivery. Four randomsamples were taken from each placenta, placed in RNAlater stabilizationsolution (Ambion, Austin, Tex.) and stored at −70′ C. RNA isolation wasperformed using Qiagen RNAeasy Maxi Kit (Qiagen, Valencia, Calif.).

We detected an increase in both placental sFlt-1 mRNA and maternal serumsFlt-1 protein in pre-eclamptic pregnant women as compared tonormotensive pregnant women. The average serum level of sFlt-1 wasalmost four times higher in the severe pre-eclampsia patients ascompared to normal control pregnant women. To exclude the possibilitythat this effect was due to the earlier gestational age of thepre-eclamptic cases, we also measured sFlt-1 levels in gestationallymatched normotensive women delivering pre-maturely for other reasons(gestational ages 23-36 weeks), and we found no significant differencein this group compared with normotensive term pregnancies. The probesused for northern blots were obtained by PCR and included a 500 bpfragment in the coding region from pUC 118 human flt-1 cDNA, and a GAPDHcDNA that was used as normalization control.

In normal pregnancy there is a balance between pro- and anti-angiogenicfactors secreted by the placenta that is necessary for adequateplacental development. We hypothesized that in pre-eclampsia, increasedproduction of sFlt-1 and decreased production of VEGF and PlGF shiftsthe balance in favor of anti-angiogenesis. To address the netanti-angiogenic activity we measured VEGF and PlGF serum levels andfound that PlGF and VEGF serum levels were lower in patients withpre-eclampsia as compared to normal control patients (mean PlGF,235.3+/−45.3 pg/ml versus 464+/−116.6 pg/ml) as has been described(Tidwell et al., Am. J. Obstet. Gynecol., 184:1267-1272, 2001). When weincorporated sFlt-1, VEGF and PlGF levels into an anti-angiogenic index,or PAAI, as an indicator of net anti-angiogenic activity, we found thatwe could clearly separate the pre-eclamptic from the normal patients andthat the PAAI seemed to correlate with severity of the pre-eclampsia(FIG. 1C). This PAAI can be used as a diagnostic tool for the detectionof pre-eclampsia in pregnant women.

EXAMPLE 2 Serum from Women with Pre-Eclampsia Inhibits Angiogenesis inan In Vitro Endothelial Tube Assay

We hypothesized that excess circulating sFlt-1 in patients withpre-eclampsia causes endothelial dysfunction and leads to ananti-angiogenic state. To address this, we used an endothelial tubeassay as an in vitro model of angiogenesis. Growth factor reducedMatrigel (7 mg/mL, Collaborative Biomedical Products, Bedford, Mass.)was placed in wells (100 μl/well) of a pre-chilled 48-well cell cultureplate and incubated at 37° C. for 25-30 minutes to allow polymerization.Human umbilical vein endothelial cells (30,000+in 300 μl of endothelialbasal medium with no serum, Clonetics, Walkersville, Md.) at passages3-5 were treated with 10% patient serum, plated onto the Matrigel coatedwells, and incubated at 37° C. for 12-16 hours. Tube formation was thenassessed through an inverted phase contrast microscope at 4× (NikonCorporation, Tokyo, Japan) and quantitatively analyzed (tube area andtotal length) using the Simple PCI imaging analysis software.

The conditions of the tube formation assay were adjusted such thatnormal human umbilical vein endothelial cells form tubes only in thepresence of exogenous growth factors such as VEGF. Under theseconditions, we found that while serum from normotensive women inducedendothelial cells to form regular tube-like structures, serum from womenwith pre-eclampsia inhibited tube formation (FIG. 2). Notably, by 48hours post-partum this anti-angiogenic effect had disappeared suggestingthat the inhibition of tubes noted with the serum from pre-eclampsiapatients was probably due to a circulating factor released by theplacenta. When sFlt-1 was added to normotensive serum at doses similarto those found in patients with pre-eclampsia, tube formation did notoccur, mimicking the effects seen with the serum from pre-eclampticwomen. When exogenous VEGF and PlGF were added to the assay usingpre-eclamptic serum, tube formation was restored (FIG. 2). Recombinanthuman VEGF, human PlGF, and human Flt-1Fc were used for these assays.These results suggested that the anti-angiogenic properties ofpre-eclamptic serum were due to the antagonism of VEGF and PlGF byendogenous sFlt-1. These results also suggested that addition ofpurified VEGF and/or PlGF can reverse or mitigate the pre-eclampticcondition and can be used therapeutically.

EXAMPLE 3 sFlt-1 Inhibits VEGF and PlGF Induced Vasodilation of RenalMicrovessels

The causative role of sFlt-1 in vasoconstriction was determined using anin vitro microvascular reactivity experiment. Microvascular reactivityexperiments were done as described previously using rat renalmicrovessels (Sato et al., J. Surg. Res., 90:138-143, 2000). Kidneyartery microvessels (70-1701 m internal diameter) were dissected fromrat kidneys using a 10× to 60× dissecting microscope (Olympus Optical,Tokyo, Japan). Microvessels were placed in an isolated microvesselchamber, cannulated with dual glass micropipettes measuring 30-60 μm indiameter, and secured with a 10-0 nylon monofilament suture (Ethicon,Somerville, N.J.). Oxygenated (95% oxygen and 5% carbon dioxide) Krebs'buffer solution warmed to 37° C. was continuously circulated through thevessel chamber and a reservoir containing a total of 100 ml of thesolution. The vessels were pressurized to 40 mmHg in a no-flow stateusing a burette manometer filled with a Krebs' buffer solution. With aninverted microscope (40× to 200×; Olympus CK2, Olympus Optical)connected to video camera, the vessel image was projected onto ablack-and-white television monitor. An electronic dimension analyzer(Living System Instrumentation, Burlington, Vt.) was used to measure theinternal lumen diameter. Measurements were recorded with a strip-chartrecorder (Graphtec, Irvine, Calif.). Vessels were allowed to bathe inthe microvessel chamber for at least 30 minutes prior to anyintervention. In all experimental groups, the relaxation responses ofkidney microvessels were examined after pre-contraction of themicrovessels with U46619 (thromboxane agonist) to 40-60% of theirbaseline diameter at a distending pressure of 40 mmHg. Once thesteady-state tone was reached, the responses to various reagents such asVEGF, PlGF, and sFlt-1 were examined. Recombinant rat VEGF, mouse PlGF,and mouse Flt-1 Fc were used for these assays. All drugs were appliedextraluminally. Measurements were made when the response had stabilized(usually 2-3 minutes after the drug was administered). One to fourinterventions were performed on each vessel. The vessels were washedwith a Krebs' buffer solution and allowed to equilibrate in a drug-freeKrebs' buffer solution for 20-30 minutes between interventions.

We found that sFlt-1 alone did not cause significant vasoconstriction,however it blocked the dose responsive increase in vasodilation inducedby VEGF or PlGF (FIG. 3A). Furthermore, we found that VEGF and PlGF, atphysiological levels seen in pregnancy, induced significant dosedependent arteriolar relaxation, and that this effect was blocked by theaddition of 10 ng/ml sFlt-1, a concentration observed in severelypre-eclamptic women (FIG. 3B). This result suggested that circulatingsFlt-1 in patients with pre-eclampsia may oppose vasorelaxation, thuscontributing to hypertension. These results support the conclusion thatsFlt-1 is responsible for many of the clinical and pathological symptomsof pre-eclampsia, including hypertension. Inhibition of sFlt-1, throughthe use of directed antibodies, for example, could reverse the effectsof the protein in pre-eclamptic women and such sFlt-1 inhibitors couldpotentially be used as a therapeutic agent.

EXAMPLE 4 Effects of sFlt-1 in an Animal Model of Pre-Eclampsia

Based on the above results, we hypothesized that the addition ofexogenous sFlt-1 would produce hypertension and proteinuria in an animalmodel. Adenovirus expressing sFlt-1 has been shown to produce sustainedsystemic sFlt-1 levels associated with significant anti-tumor activity(Kuo et al., Proc. Natl. Acad. Sci. USA, 98:4605-4610, 2001). Thisrecombinant adenovirus encoding murine sFlt-1 was injected into the tailvein of pregnant Sprague-Dawley rats on day 8-9 of pregnancy. Adenovirusencoding murine Fc and sFlk1-Fc (fusion protein of mouse VEGF receptor 1Flk1 ectodomain and Fc protein) in equivalent doses were used ascontrols. Flk1 has been shown to bind to VEGF, but not PlGF. Hence,sFlk-1 Fc was chosen as a control to help discriminate between theanti-VEGF and the anti-PlGF activity of sFlt1.

Both pregnant and non-pregnant Sprague-Dawley rats were injected with1×10⁹ pfu of Ad Fc, Ad sFlt-1, or Ad sFlk-1 Fc by tail vein injections.These adenoviruses have been described previously (Kuo et al., supra)and were generated at the Harvard Vector Core Laboratory. Pregnant ratswere injected with the adenoviruses at day 8-9 of pregnancy (earlysecond trimester) and blood pressure was measured at day 16-17 ofpregnancy (early third trimester). In non-pregnant animals, BPs weremeasured at day 8 after injection of the adenoviruses. BPs were measuredin the rats after anesthesia with pentobarbital sodium (60 mg/kg, i.p.).The carotid artery was isolated and cannulated with a 3-Fr high-fidelitymicrotip catheter connected to a pressure transducer (MillarInstruments, Houston, Tex.). The Millar Mikro-Tip catheter was advancedinto the artery to record blood pressure. Blood pressure and heart ratewere recorded in by chart-strip recorder (model 56-1×40-006158, GouldInstrument Systems, Cleveland, Ohio) and averaged over a 10-minuteperiod. Blood, tissue, and urine samples were then obtained beforeeuthanasia. Urinary albumin was measured by standard dipstick andquantitated by competitive enzyme-linked immunoassay (ELISA) as has beendescribed elsewhere (Cohen et al., Kidney Intl., 45:1673-1679, 1994).Urinary creatinine was measured by a picric acid colorimetric procedurekit (Sigma, St. Louis, Mo.). We measured intrarterial blood pressures inthe early third trimester of the pregnancy to mimic the naturalpathology of pre-eclampsia. These experiments were also performed innon-pregnant female Sprague-Dawley rats to determine if the effects ofsFlt-1 is direct or indirect through its effects on the placenta.Systemic levels of sFlt-1 on the day of blood pressure measurement wereconfirmed by Western blot analysis to be in the range of 25-350 ng/mL inthe various sFlt-1 treated animals on the day of BP measurements. Bloodpressure and proteinuria in the different experimental groups is shownin Table 1.

TABLE 1 Blood Pressure and Proteinuria in Rats N MAP (mmHg) U alb:crratio Fc (P) 5 75.6 ± 11.1 62 ± 21 sFlt-1 (P) 4 109.0 ± 19.3* 6923 ±658* sFlk-1Fc (P) 4 72.8 ± 14.7 50 ± 32 Fc (NP) 5 89.3 ± 5.7  138 ± 78 sFlt-1 (NP) 6 117.9 ± 12.9* 12947 ± 2776* sFlk-1Fc (NP) 4 137.3 ± 2.3* 2269 ± 669* Pregnant (P) and nonpregnant (NP) rats were administeredadenovirus expressing Fc (control), sFlt-1, or sFlk-1Fc protein. Meanarterial blood pressure (MAP = diastolic + ⅓ pulse pressure in mmHg) ±S.E.M and urine albumin:Cr ratio (mg of albumin per gram of creatinine)± S.E.M were measured eight days later, corresponding to the early thirdtrimester in the pregnant rats. N = the number of animals in eachexperimental group. The * represents statistical significance with p <0.01 when compared with the control group (Fc).

Pregnant rats treated with sFlt-1 had significant hypertension andnephrotic range albuminuria compared with Fc controls. Nonpregnant ratsadministered sFlt1 also developed hypertension and proteinuria. Notably,the sFlk-Fc treated nonpregnant rats developed hypertension andproteinuria, whereas the sFlk-Fc treated pregnant rats did not. Inpregnancy, therefore, the antagonism of VEGF alone is insufficient toproduce pre-eclampsia, possibly due to the presence of high levels ofPlGF. In the nonpregnant state, where PlGF is virtually absent,antagonism of VEGF alone is sufficient to disrupt thepro/anti-angiogenic balance and produce renal pathologies similar tothose associated with pre-eclampsia. Various staining techniques wereused to examine the renal lesion that was observed in all sFlt-1 treatedrats (FIG. 4). Harvested kidneys from the rats were fixed in Bouin'ssolution, sectioned and stained with H&E and PAS stains. For electronmicroscopy, renal tissue was fixed in glutaraldehyde, embedded inaraldite-epon mixture, and ultrathin kidney sections (1 μm) were cut,stained with Toluene blue and assessed using a Zeiss EM 10 at variousmagnifications. Immunofluorescence for fibrin deposits within theglomeruli was done using polyclonal anti-fibrin antibody (ICN,Switzerland). Global and diffuse glomerular endotheliosis was the renallesion universally observed in the sFlt-1 treated rats. We detectedglomerular enlargement with occlusion of the capillary loops by swellingand hypertrophy of endocapillary cells. Numerous apparent proteinresorption droplets were seen in the glomerular epithelial cells. Nosegmental glomerulosclerosis was observed. Isolated “double contours”and focal deposition of fibrin within the glomeruli were seen. Thisfinding of fibrin deposition in the absence of significant mesangialinterposition is similar to what has been described as typical of thepre-partum stage of the human disease (Kincaid-Smith, Am. J. KidneyDis., 17:144-148, 1991). Immunofluorescence for fibrin showed foci offibrin deposition within the glomeruli of sFlt-1 treated animals but notFc treated animals. The sFlk1 treated nonpregnant rats developed thesame lesion. In fact, when sFlk 1 was used at the same levels as sFlt-1,the renal damage was more severe in the non-pregnant rats, as there arefewer circulating pro-angiogenic molecules for the sFlt-1 to antagonize.These results suggested that elevated levels of sFlt-1 may beresponsible for the glomerular endotheliosis associated withpre-eclampsia, but that this effect was independent of the placentasince glomerular changes were detected in nonpregnant as well aspregnant rats. These results also suggested that antagonism of both VEGFand PlGF is important in the pathology of pre-eclampsia as hypertensionand proteinuria occurred in sFlk-1 treated non-pregnant mice but not insFlk-1 treated pregnant mice where PlGF levels are high.

The animal model created herein can be used as an experimental model totest novel therapeutic compounds. Both the efficacy of potentialtherapeutic compounds and the pharmacology and toxicity can be studiedusing this animal model.

EXAMPLE 5 Effects of sFlt-1 in an Animal Model of Eclampsia

Pregnant rats in their early second trimester of pregnancy are injectedwith exogenous sFlt-1. The rats are then monitored and tested duringtheir early third trimester for the development of eclampsia. Tests usedfor detection of eclampsia can include MRI of the rat brains for thedevelopment of edema, EEG of the rat brain for the development ofseizures, and histology of the rat brains to determine if endothelialdamage has occurred along the blood-brain barrier and choroids-plexususing specific endothelial markers.

The animal model created herein can be used as an experimental model totest novel therapeutic compounds. Both the efficacy of potentialtherapeutic compounds and the pharmacology and toxicity can be studiedusing this animal model.

EXAMPLE 6 PlGF/Creatinine Ratio in Urine is Diagnostic of Pre-Eclampsia

Urine samples were obtained from 10 women at 16 weeks gestation (fivenormals, four mild preeclamptics, and one severe pre-eclamptic). Thesesamples were provided by Dr. Ravi Thadhani at Massachusetts GeneralHospital. The average urinary free PlGF/creatinine ratios (pg PlGF permg of creatinine) for the normal pregnant women were 78+/−10.7 and forthe four mild pre-eclamptics were 33+/−5.0 and for the one severepreeclamptic patient was 17. Thus, an alteration in the ratio of PlGF tocreatinine in urine is useful as a diagnostic indicator forpre-eclampsia in a patient.

EXAMPLE 7 Urinary PlGF Levels Measure in Control and Pre-EclampticPregnant Women

Urinary PlGF was measured in control pregnant women and pre-eclampticwomen using archived urine specimens from the CPEP trial (see Example 8)in collaboration with Dr. Richard Levine at the NIH (Table 2). The tablebelow shows significant decreases in urinary PlGF in patients who laterdeveloped pre-eclampsia during mid-pregnancy (22-30 weeks) and latepregnancy (>30 weeks), but not in early pregnancy (<20 weeks). All urinespecimens were obtained prior to clinical symptoms of pre-eclampsia.

TABLE 2 Urinary PIGF levels in pg/ml in pre-eclamptic versus controlpregnant patients. Control (n = 118) PE (n = 120) Early pregnancy 39.8042.28 Mid pregnancy 193.11 98.66 (p < 0.0001) Late pregnancy 107.8262.05 (p = 0.0213)

EXAMPLE 8 sFlt-1 and PlGF Protein Levels as a Diagnostic Indicator ofPre-Eclampsia and Eclampsia in Women

For this study we used archived samples from the Calcium forPre-eclampsia Prevention trial in order to analyze the gestationalpatterns of circulating sFlt-1, free PlGF, and free VEGF in normotensiveand pre-eclamptic pregnancies in collaboration with Dr. Richard Levineat the NIH. Calcium for Pre-eclampsia Prevention, or CPEP, was arandomized, double-blind clinical trial conducted during 1992-1995 toevaluate the effects of daily supplementation with 2 grams elementalcalcium or placebo on the incidence and severity of pre-eclampsia(Levine et al., N. Engl. J. Med. 337:69-76, 1997; Levine et al., ControlClin. Trials 17:442-469, 1996). Healthy nulliparous women with singletonpregnancies were enrolled between 13 and 21 weeks gestation at fiveparticipating U.S. medical centers and followed until 24 hourspostpartum using a common protocol and identical data collection forms.At enrollment, all CPEP participants had blood pressure <135/85 mm Hg,and none had renal dysfunction or proteinuria. Gestational age wasdetermined by ultrasound examination. Serum specimens were obtained fromparticipants prior to enrollment in the trial (13-21 weeks), at 26-29weeks, at 36 weeks if still pregnant, and when hypertension orproteinuria were noted. “Endpoint specimens” (active PE) were specimensobtained at or after onset of pre-eclampsia symptoms and signs, butbefore labor and delivery as described elsewhere (Levine et al., 1996,supra). Archived blood samples from the CPEP trial were obtained throughcollaboration with Dr. Richard Levine at the NIH.

Participants

We selected subjects having complete outcome information, serum samplesobtained at <22 weeks, and a liveborn male infant. Of 4,589 CPEPparticipants, we excluded 253 lost to follow-up, 21 whose pregnancy hadterminated prior to 20 weeks, 13 missing maternal or perinatal outcomedata, 4 without smoking history, 9 with hypertension not verified bychart review teams, and 32 others with stillbirths, leaving 4,257 womenwith adequate information and live births. Among these 2,156 had maleinfants. After excluding one woman whose infant had a chromosomalabnormality, 381 with gestational hypertension, and 43 without abaseline serum specimen, 1,731 women remained. Of these, 175 developedpre-eclampsia and 1,556 remained normotensive throughout pregnancy.

Since calcium supplementation had no effect on the risk and severity ofpre-eclampsia and was unrelated to concentrations of pro- andanti-angiogenic molecules, cases and controls were chosen without regardto CPEP treatment. For each pre-eclampsia case one normotensive controlwas selected, matched for enrollment site, gestational age at collectionof the first serum specimen (within one week), and freezer storage timeat −70° C. (within 12 months). 120 matched pairs (“cases” and“controls”) were randomly chosen for analysis of all 657 serum specimensobtained before labor (Table 3, below). Mean gestational age atcollection of the first serum specimen was 112.8 and 113.6 days in casesand controls, respectively; mean duration of freezer storage was 9.35and 9.39 years.

TABLE 3 Characteristics of cases and controls at CPEP enrollment and oftheir newborn infants Cases Controls Characteristic (n = 120) (n = 118)Age (yr)  20.8 ± 4.5  20.0 ± 3.4 Body mass index  27.3 ± 6.8  25.0 ±6.1** Systolic blood pressure (mm Hg) 109.0 ± 9.0 106.0 ± 9.0† Diastolicblood pressure (mm Hg)  62.0 ± 8.0  59.0 ± 7.0‡ Gestational age atdelivery (wks)  38.1 ± 2.6  38.9 ± 2.5* Current smoker [n (%)]  9 (7.5)13 (11.0) Ever married [n (%)] 25 (20.8) 22 (18.6) Race/ethnicity^(•)White, non-Hispanic [n (%)] 24 (20.0) 33 (28.0) White, Hispanic [n (%)]21 (17.5) 14 (11.9) African-American [n (%)] 69 (57.5) 68 (57.6) Other,unknown [n (%)]  6 (5.0)  3 (2.5) Birthweight (g)  3100 ± 796 3247 596Delivery <37 wks [n (%)] 26 (21.7)  9 (7.6)§ Small for gestational age18 (15.0)  4 (3.4)§ (<10^(th) percentile) [n (%)] Mean ± standarddeviation unless indicated P = 0.03 **P = 0.007 †P = 0.001 ‡P = 0.006 §P= 0.002 ^(•)race or ethnic group was self-reported.

For this study, hypertension was defined as a diastolic blood pressureof at least 90 mm Hg on two occasions 4-168 hours apart. Severehypertension was defined as a diastolic blood pressure of at least 110mm Hg on two occasions 4-168 hours apart, or one occasion if the womanhad received anti-hypertensive therapy. Proteinuria was defined as 300mg or more protein in a 24-hour urine collection, two random urinespecimens 4-168 hours apart containing at least 1+ protein by dipstick,a single urine sample with a protein/creatinine ratio at least 0.35, ora single random urine specimen containing at least 2+ protein bydipstick. Severe proteinuria was diagnosed by a 24-hour urine collectionsample containing at least 3.5 g protein or by two random urinespecimens with at least 3+ protein by dipstick. Pre-eclampsia wasdefined as hypertension and proteinuria occurring within 7 days of eachother; severe pre-eclampsia was defined as pre-eclampsia with severehypertension, severe proteinuria, HELLP syndrome (hemolysis, elevatedliver enzymes, low platelets), or eclampsia. The onset of pre-eclampsiawas the time of detection of the first elevated blood pressure orproteinuria in the urine sample leading to the diagnosis ofpre-eclampsia.

Small for gestational age (SGA) was defined as birth weight lower thanthe 10^(th) percentile for gestational age according to US tables ofbirth weight for gestational age by race, parity, and infant sex (Zhangand Bowes 1995, supra).

Procedures

Assays were performed at the Beth Israel Deaconess Medical Center bylaboratory personnel who were blinded to patients' diagnoses and otherrelevant clinical information. Specimens were randomly ordered foranalysis. Enzyme-linked immunosorbent assays (ELISA) for human sFlt-1,free PlGF, and free VEGF were performed according to the manufacturer'sinstructions, using kits purchased from R&D Systems (Minneapolis,Minn.). Aliquots of serum samples which had been stored at −70° C., werethawed to room temperature, diluted with BSA/Tris-buffered saline, andincubated for 2 hours in a 96-well plate pre-coated with a captureantibody directed against sFlt-1, PlGF, or VEGF. The wells were thenwashed three times, incubated 20 minutes with a substrate solutioncontaining hydrogen peroxide and tetramethylbenzidine, and the reactionquenched with 2N sulfuric acid. Optical density was determined at 450 nm(wavelength correction 550 nm). All assays were performed in duplicate.Protein concentrations were calculated using a standard curve derivedfrom known concentrations of the respective recombinant proteins. If thedifference between duplicates exceeded 25%, the assay was repeated andinitial results discarded. The assays had sensitivities of 5, 7, and 5pg/ml for sFlt 1, PlGF, and VEGF, respectively, with inter- andintra-assay coefficients of variation of 7.6% and 3.3% for sFlt 1, 11.2%and 5.4% for PlGF, and 7.3% and 5.4% for VEGF.

Statistical Analysis

Chi-square and t tests were used in analyses of maternal or infantcharacteristics to compare categorical or continuous variables,respectively. Although arithmetic mean values of concentrations aregiven in text and figures, statistical testing was performed afterlogarithmic transformation unless noted otherwise. Adjustment wasperformed using logistic regression on logarithmically transformedconcentrations.

Results

Of the 120 cases, 80 developed mild and 40 severe pre-eclampsia,including 3 with HELLP syndrome and 3 with eclampsia. Case patients wereshorter than control patients, had a higher body mass index, and higherbaseline blood pressure (Table 2). In addition, larger proportions ofcase patients had pregnancies complicated by pre-term delivery orsmall-for-gestational age (SGA) infants. Case patients contributed anaverage of 2.9 serum specimens to the study; controls, 2.6 specimens.

We first confirmed that sFlt-1, PlGF, and VEGF were altered in patientswith pre-eclampsia at the time of active disease as compared togestationally matched controls from this CPEP study group. Specimensdrawn at the time of established clinical pre-eclampsia (endpointspecimens) had dramatically increased sFlt-1 levels, decreased PlGFlevels, and decreased VEGF levels compared to controls with gestationalages (4382 vs. 1643 pg/ml sFlt1, p<0.0001; 137 vs. 669 pg/ml PlGF,p<0.0001; and 6.41 vs. 13.86 pg/ml VEGF, p=0.06) for cases and controls,respectively, in 23 gestational-age matched pairs) similar to priorpublished reports (Maynard et al., J. Clin. Invest. 111:649-658, 2003).

In order to evaluate the gestational pattern of sFlt-1, PlGF and VEGFlevels, we measured circulating concentrations of sFlt-1, PlGF, and VEGFfrom serum specimens obtained from case patients and control patientswithin various gestational age windows. The gestational pattern ofsFlt-1 protein for 120 pre-eclamptic and 120 control women is shown inFIG. 5A. sFlt-1 levels in control patients remained constant until 33-36weeks, when they rose by approximately 145 pg/ml per week until laborand delivery. Among case patients before clinical symptoms, sFlt-1appeared to begin to rise at 21-24 weeks, with a steeper rise and astatistically significant difference from controls at 29-32 weeks (FIG.5A). Overall, differences between case and control patients measuredbefore the onset of clinical symptoms were 17% (p<0.05) atmid-gestation. The end-point specimens were significantly elevated ascompared to specimens drawn prior to the disease. In order to evaluatethe mechanisms of sFlt-1 rise prior to the onset of clinical disease, weplotted sFlt-1 concentrations on all pre-eclamptics by weeks prior tothe onset of pre-eclampsia (FIG. 5B). Mean sFlt-1 concentrations inspecimens from case patients were plotted by completed weeks beforeonset of pre-eclampsia. Beginning at 5 weeks prior to pre-eclampsia,sFlt-1 concentrations rose substantially until 1 week prior to the onsetof disease when they approached the concentrations observed in endpointspecimens. The increases in sFlt-1 at 4, 3, 2, and 1 week(s) beforepre-eclampsia occurred with little change in mean gestational age andcannot be explained by late third trimester increases with advancinggestational age. From 8-6 to 5 weeks before pre-eclampsia sFlt-1increased 962 pg/ml, while mean gestational age rose 31 days. Aboutone-third of this increase in sFlt-1 cannot be attributed to advancinggestation. When sFlt-1 was graphed by gestational age in controls and incases after removing specimens obtained ≦5 weeks before onset ofpre-eclampsia, no substantial differences were observed (FIG. 5C). Thesedata suggest that the higher sFlt-1 concentration in case patients priorto onset of pre-eclampsia is due to acute rises in sFlt-1 within the 5weeks before onset of clinical disease.

We then plotted the gestational pattern of PlGF protein in the samepatient group as shown in FIG. 6A. Control PlGF protein concentrationsrose during the first two trimesters, peaked at 29-32 weeks, and fellduring late gestation. Among case patients, prior to pre-eclampsia, PlGFprotein concentrations followed a similar gestational pattern, but weresignificantly lower than controls from 13-16 weeks. Overall, differencesin PlGF between cases patients and controls measured before the onset ofclinical symptoms were 35% (p<0.0001) at mid-gestation. PlGF levels incases prior to onset of pre-eclampsia is depicted by weeks beforepre-eclampsia (FIG. 6B), and by gestational age after removing specimens<5 weeks before pre-eclampsia (FIG. 6C). By 1 week prior to onset ofpre-eclampsia, concentrations approached those observed after onset ofpre-eclampsia (FIG. 6B). Compared to controls, PlGF levels from casepatients were moderately reduced remote from delivery, with moresubstantial reductions at 5 and 3 weeks before delivery. Concentrationsfrom control patients remained high from 17-15 through 3 weeks beforedelivery, then fell dramatically. The graph showing PlGF levelsexcluding specimens obtained ≦5 weeks before pre-eclampsia indicates asmaller decrease in cases relative to controls at 29-32 weeks ofgestation and none at all in specimens obtained from case patients at33-36 weeks (FIG. 6C). This suggests that the fall in PlGFconcentrations in the weeks prior to the disease was responsible for thedramatically low levels of PlGF noted at the onset of disease (or endpoint specimens shown in FIG. 6A).

VEGF concentrations throughout pregnancy were very low and similar incontrols and cases before pre-eclampsia, except for a significantdecrease in case patients at 37-41 weeks. Mean VEGF concentrations at23-32 weeks in cases excluding specimens obtained 5 weeks beforepre-eclampsia did not differ significantly from controls (11.6 vs. 12.8pg/ml), whereas concentrations in cases including specimens ≦5 weeksbefore delivery did (5.1 vs. 12.8 pg/ml, p<0.01). At 33-41 weeks caseVEGF concentrations >5 or ≦5 weeks before pre-eclampsia were higher andlower than controls, respectively (11.2 pg/ml and 8.3 vs. 9.7 pg/ml),although these differences were not significant.

FIG. 7 depicts sFlt-1 and PlGF at 23-32 weeks (FIG. 7A) and 33-41 weeks(FIG. 7B) by pre-eclampsia status and severity. The graphs show thatsFlt-1 increases and PlGF decreases before onset of pre-eclampsia wereassociated with disease severity, time of onset, and the presence of anSGA infant. At 23-32 weeks, sFlt-1 and PlGF in case patients with an SGAinfant before onset of pre-eclampsia were significantly higher or lower,respectively, than corresponding concentrations in control patients withan SGA infant. Moreover, in comparison to control patients who deliveredpre-term, case patients with pre-term delivery had higher sFlt-1 andsignificantly lower PlGF.

To determine whether concentrations of sFlt-1 or PlGF prior to clinicalsigns of pre-eclampsia were associated with the risk of this condition,we calculated odds ratios for pre-eclampsia for each quartile of controlvalues of sFlt-1 and PlGF, as compared to the lowest or highestquartile, respectively (Table 4). We also examined the pre-eclampsiarisk of the extreme quartiles with respect to all other quartiles, asfollows. For specimens obtained in the second- and earlythird-trimester, the lowest quartile of PlGF was associated with anincreased risk of preterm (<37 weeks gestation) pre-eclampsia (OR 7.4,95% CI 1.8 to 30.2 for 13-20 week specimens; OR 7.9, 95% CI 2.9 to 21.5for 21-32 week specimens). A level of PlGF in the lowest quartile,however, was not a significant predictor of term (≧37 weeks)pre-eclampsia. For sFlt-1, associations with pre-eclampsia were observedonly closer to disease onset. An sFlt-1 level in the highest quartilebetween 21 to 32 weeks gestation (but not earlier) predicted pretermpre-eclampsia 5 (OR 5.1, 95 percent CI 2.0 to 13.0), and a level in thehighest quartile between 33 and 41 weeks (but not earlier) predictedterm pre-eclampsia (OR 6.0, 95 percent CI 2.9 to 12.5). This isconsistent with FIG. 5B, which shows that elevation of sFlt-1 occurslargely within 5 weeks of onset of clinical disease. The lowest quartileof VEGF was not predictive of pre-eclampsia.

TABLE 4 Odds Ratios (OR) for Pre-eclampsia at <37 and ≧37 Weeks Prior toClinical Signs by Quartiles of Total sFlt-1 and Free PIGF in Controls at13-20, 21-32, and 33-41 Weeks Gestation sFlt-1 PE < 37 wks PE ≧ 37 wksPIGF PE < 37 wks PE ≧ 37 wks (pg/ml) Controls N N OR* N OR* (pg/ml)Controls N N OR* N OR* 13-20 wks Q4: >1047 25 6 1.3 (0.4-5.0) 20 1.5(0.6-3.7) Q4: >307 25 4  1.0 Referent  4 1.0 Referent Q3: >698-1047 25 82.2 (0.6-7.8) 23 1.9 (0.8-4.5) Q3: >160-307 25 2  0.6 (0.1-3.5) 22 5.6(1.7-19.0) Q2: >531-698 25 4 0.5 (0.1-2.3) 16 1.1 (0.4-2.7) Q2: >87-16025 6  1.9 (0.4-8.2) 26 6.4 (1.9-22.1) Q1: ≦531 25 6 1.0 16 1.0 ReferentQ1: ≦87 25 12  9.6 (1.6-57.6) 23 6.7 (1.6-27.5) Referent 21-32 wksQ4: >1131 25 16 4.7 (1.3-16.6) 18 1.7 (0.7-4.4) Q4: >1021 25 1  1.0Referent 14 1.0 Referent Q3: >743-1131 26 5 1.4 (0.3-6.0) 21 1.7(0.7-4.2) Q3: >677-1021 26 1  1.1 (0.1-18.2) 19 1.2 (0.5-3.1)Q2: >512-743 25 1 0.3 (0.0-2.8) 21 1.9 (0.8-4.7) Q2: >363-677 25 5  5.3(0.6-49.3) 20 1.3 (0.5-3.2) Q1: ≦512 26 4 1.0 14 1.0 Referent Q1: ≦36326 19 19.6 (2.3-163.8) 21 1.2 (0.5-3.1) Referent sFlt-1 PIGF (pg/ml)Controls N PE < 37 wks PE ≧ 37 wks (pg/ml) Controls N PE < 37 wks PE ≧37 wks 33-41 wks Q4: >2191 22 44 7.5 (2.6-21.8) Q4: >948 22 6 1.0Referent Q3: >1633-2191 22 12 1.7 (0.5-5.5) Q3: >377-948 22 18 2.7(0.9-8.3) Q2: >1287-1633 22 7 1.0 (0.3-3.3) Q2: >175-377 22 19 2.8(0.9-8.5) Q1: ≦1287 23 8 1.0 Referent Q1: ≦175 23 28 4.1 (1.4-12.2)*Odds ratio adjusted for gestational age and body mass index (with 95%CI). OR with 95% CI > 1.0 in bold type. Case specimens were obtainedprior to clinical signs of pre-eclampsia.

These results demonstrate that sFlt-1 levels begin to rise dramaticallyabout 5 weeks before the onset of pre-eclampsia symptoms. Parallel withthe rise in sFlt-1, free PlGF and free VEGF levels fall, suggesting thatthe decrease in PlGF and VEGF may be due at least partially toantagonism by sFlt-1 and not due to a decrease in placental productionof PlGF and VEGF. Three pre-eclampsia subgroups—severe pre-eclampsia,early onset of disease, and SGA infants—had higher sFlt-1 and lower PlGFconcentrations at 23-32 weeks and at 33-41 weeks than controls or womenwith mild pre-eclampsia. We have also demonstrated a small butsignificant decrease in free PlGF beginning early in the secondtrimester among women destined to develop pre-eclampsia. These resultsdemonstrate that a decrease in PlGF levels may be a useful predictor ofearly onset pre-eclampsia.

We describe here for the first time the gestational pattern of sFlt-1 innormal pregnancy, observing relatively stable levels throughoutgestation followed by a steady increase beginning at 33-36 weeks. Thisrise corresponds to the late gestational fall in PlGF observed in normalpregnancy by others (Torry et al., J. Soc. Gynecol. Invest. 10:178-188,1998; Taylor et al., Am. J. Obstet. Gynecol. 188:177-182, 2003) and inthe results described herein. The temporal association, together withthe knowledge that sFlt-1 interferes with PlGF ELISA measurement(Maynard et al., supra) suggests that the fall in free PlGF levelsduring late gestation may be due to the rise in sFlt-1 levels. Duringfirst and second trimesters, when placental growth is needed to keeppace with increasing fetal demands, PlGF concentrations are high andsFlt-1 concentrations are low, creating a relatively pro-angiogenicstate. Later in gestation, when placental vascular growth may need to betempered and halted, there is a rise in the anti-angiogenic sFlt-1 andresulting decrease in PlGF. In women with pre-eclampsia, the sFlt-1 risebegins earlier in gestation, approximately five weeks before symptomonset, at about 29-32 weeks gestation on average. Thus, inpre-eclampsia, the anti-angiogenic “brakes” may be applied too soon andtoo strongly, resulting in an exaggeration of a normal physiologicprocess which arrests placental growth. It seems clear that thepathologic placental changes that characterize pre-eclampsia occur earlyin gestation (10-14 weeks), well before the dramatic rise in sFlt-1. Theresulting placental ischemia itself may enhance sFlt-1 production,ultimately triggering a burst in sFlt-1.

In addition to the large differences seen in the five weeks prior to thedevelopment of clinical symptoms, women destined to developpre-eclampsia had small, but statistically significant, decreases infree PlGF as early as 13-16 weeks gestation. This fall in PlGF generallywas not accompanied by a reciprocal increase in sFlt-1 levels. However,there was a tendency towards slightly higher sFlt-1 levels in casesduring the first trimester though it was not statistically significant(For example at the 17-20 week window, average sFlt-1 levels in caseswere 865.77 pg/ml vs. 795.25 in controls). This decrease in PlGF levelsearly on in gestation might reflect a smaller placental production ofPlGF in pregnancies compromised by conditions such as pre-eclampsia orSGA. Importantly, in patients with pre-eclampsia complicated by SGA, wefound a statistically significant increase in both sFlt-1 elevation andPlGF fall prior to the disease presentation. It is also possible thatthere is no change in placental production of PlGF in pre-eclamptics andthat elevation of local sFlt-1 levels in the placenta may contribute tothe decrease in circulating free PlGF. This is supported by the findingthat placental PlGF, measured by immunohistochemistry, is not altered inpre-eclampsia (Zhou et al., Am. J. Pathol. 160:1405-1423, 2002).

In summary, we have shown that sFlt-1 starts rising in pre-eclampsia atleast 5 weeks before the onset of clinical disease which is accompaniedby decreases in circulating free PlGF and free VEGF. Decreased PlGFduring the first trimester may serve as a predictor of pre-eclampsia andelevated sFlt-1 may serve as a predictor of proximity to clinicaldisease. This data in conjunction with the animal work described abovedemonstrating sFlt-1 alone induces pre-eclampsia like symptoms inrodents suggests a probable etiological role for sFlt-1 in thepathogenesis of pre-eclampsia. Our limited data on SGA infants andpreterm delivery in controls, as compared to case patients, suggest thatthe increased alterations in protein levels observed in pre-eclampticpregnancies with an SGA infant are more substantial than a differencedue only to intrauterine growth restriction or pre-term delivery in theabsence of pre-eclampsia.

EXAMPLE 9 sFlt-1 Protein and Protein Fragments Detected in Monocytesfrom Normal and Preeclamptic Patients

Peripheral blood mononuclear cells, rich in monocytes, were isolatedfrom normal and pre-eclamptic patients and used to measure the leves ofsFlt-1 and sFlt-1 fragments. Protein extracts were prepared from thePBMCs and Flt-1/sFlt-1 levels were analyzed by Western blots using anantibody that recognizes the N-terminus of Flt-1 protein (a regioncommon to both proteins). The results of this experiment showedincreased Flt-1 and sFlt-1 levels in the monocytes from pre-eclampticpatients (FIG. 8). In addition several bands were detected that had afaster migration than full-length sFlt-1. These faster migrating bandsmay be degradation products, alternatively spliced isoforms, enzymaticcleavage products, or other forms of sFlt-1.

EXAMPLE 10 sFlt-1 Protein Levels as a Diagnostic Indicator ofCardiovascular Conditions in Women with a History of Pre-Eclampsia

Women with a history of pre-eclampsia have been shown to have apropensity to develop cardiovascular conditions (see for exampleKestenbaum et al., Am. J. Kidney Dis. 42:982-989, 2003). Given ourdiscovery of the use of s-Flt-1 as a diagnostic indicator ofpre-eclampsia or eclampsia or a predisposition to pre-eclampsia oreclampsia, a study was performed to determine if sFlt-1 could also beused as a diagnostic indicator of a propensity to develop cardiovascularconditions or events in women who have a history of pre-eclampsia. Theresults of this study are shown in Table 5.

We examined 29 normotensive women with a history of pre-eclampsia and 32normotensive women with previous normal pregnancies at 18.0±9.7 monthspostpartum in the General Clinical Research Centers at the MassachusettsInstitute of Technology in collaboration with Dr. Ravi Thadhani at theMassachussetts General Hospital. Since pre-eclampsia often presents nearterm and other disorders can lead to preterm delivery, to preventmisclassification of pregnancy outcome, all normotensive women haddelivered at term (>38 weeks). Women with current pregnancy, diabetes ora history of gestational diabetes, chronic hypertension, proteinuria orserum creatinine >1.0 mg/dL were excluded. After providing writteninformed consent, subjects underwent a history and physical examinationand a urine pregnancy test. Blood was collected on the morning after anovernight fast for measurement of free VEGF and sFlt-1. Samples wereprocessed immediately, stored at −80° C. for no longer than 18 monthsand were thawed only for the current study. Commercial assay ELISA kitswere used for sFlt-1 and free VEGF (R&D systems, Minnesota USA). Theintra- and inter-assay co-efficent of variance (CVs) for sFlt-1 and VEGFwere 3.5 and 5.6, and 8.1 and 10.9, respectively. All samples were runin duplicate by technicians blinded to pregnancy outcome.

Univariate comparisons between the pregnancy outcome groups wereperformed using two-sample t tests, Wilcoxon rank sum test or Fisherexact test as appropriate. Logistic regression was used to calculateodds ratios for having had prior pre-eclampsia given levels ofpostpartum markers, and to adjust for potential confounding.

TABLE 5 Postpartum sFlt-1 data according to pregnancy outcome.Pre-eclampsia Normotensive N = 29 N = 32 P Age (years) 33.7 ± 5.8 30.7 ±7.1 0.08 Race (% Caucasian) 86 84 0.6 Months postpartum 18.0 ± 10  18.0± 10  1.0 Body mass index (kg/m²) 29.2 ± 7.8 25.0 ± 5.8 0.02 Systolicblood pressure (mmHg) 111 ± 10 105 ± 8  0.01 Diastolic blood pressure 73 ± 10 68 ± 7 0.04 (mmHg) Mean arterial blood  86 ± 10 81 ± 7 0.01pressure (mmHg)* Oral or subcutaneous 31 34 0.8 contraception (%)Fasting glucose (mg/dL) 81 ± 7 80 ± 6 0.5 Soluble fms-like tyrosine 41.6± 6.7  30.4 ± 10.2 <0.01 kinase (pg/ml) Continuous variables arereported as mean ± standard deviation or median (interquartile range) asappropriate.

These results indicate that women with a history of pre-eclampsia duringpregnancies showed elevated levels of sFlt-1 for an extended period oftime after the pregnancy. Given that statistical analysis has shown thatwomen with a history of pre-eclampsia or eclampsia have a predispositionto develop cardiovascular conditions, these results provide support forthe use of sFlt-1 post-partum levels as a diagnostic indicator of acardiovascular condition or a propensity to develop a cardiovascularcondition.

EXAMPLE 11 Urine PlGF Levels as a Diagnostic Indicator of Pre-Eclampsia

In situations where obtaining serum measurements of VEGF, sFLT-1, andPlGF are not optimal, an alternative and less invasive screening methodmay be to measure these proteins in urine. While sFlt1 is too large amolecule (110 kDa) to be filtered into the urine in the absence ofproteinuria, PlGF and VEGF, much smaller proteins (˜30 kDa and 45 kDarespectively), are readily filtered. Unlike urinary PlGF, which isderived entirely from circulating blood, the major sources of urinaryVEGF are cells of the kidney itself: glomerular podocytes and tubularcells. Thus, urinary VEGF is unlikely to reflect the circulatingangiogenic state. We used archived urine samples to test the hypothesisthat urinary PlGF is reduced well before the onset of hypertension andproteinuria and predicts pre-eclampsia.

Participants and Specimens

Serum and urine specimens were requested from participants of the CPEPclinical trial (see Example 8) before enrollment in the trial, at 26-29weeks of gestation, at 36 weeks if they were still pregnant, and whenhypertension or proteinuria was noted. Both first morning and 24-hoururine specimens were requested; if neither was available, a random or“spot” urine specimen was collected. 24-hour urines were requested frompatients suspected of pre-eclampsia. “End-point specimens” referred tothose obtained at or after the onset (defined below) of signs ofpre-eclampsia, but before labor and delivery.

For the present study, we selected women with complete outcomeinformation, serum samples obtained at less than 22 weeks of gestation,and a live-born male infant. This group had previously been selected fora study of fetal DNA and pre-eclampsia, in which fetal and maternal DNAwere differentiated through the amplification of a gene on the Ychromosome. Analysis of previous work revealed no significantdifferences in maternal serum sFlt1 or PLGF concentrations according toinfant gender.

Since calcium supplementation had no effect on the risk or severity ofpre-eclampsia (Levine et al., supra) or on the concentrations ofangiogenic factors in serum (Levine et al., N. Engl. J. Med.350:672-683, 2004) or urine, women were chosen without regard to whetherthey had received calcium supplementation or placebo. For each womanwith pre-eclampsia, one normotensive control was selected, matchedaccording to enrollment site, gestational age at the collection of thefirst serum specimen, and storage time of the samples at −70° C. A totalof 120 matched pairs were randomly chosen for analysis of all serum andurine specimens obtained before labor. If a woman had more than oneurine specimen obtained on the same day, we selected one specimen,preferring first morning to random urine and random to 24-hour urine. Weidentified 348 urine specimens from 120 pre-eclampsia cases and 318 from118 normotensive controls. Two normotensive controls from the serumstudy had no eligible urine specimens and were excluded from furtheranalyses.

We examined separately urine samples obtained at 21-32 weeks ofgestation from controls and cases with onset of pre-eclampsia beforeterm (<37 weeks) for which a serum specimen from the same woman had beencollected within 3 days. There were a total of 89 urine-serum specimenpairs from 20 cases of preterm pre-eclampsia and 69 normotensivecontrols.

Pre-eclampsia was defined as described above. The time of onset ofpre-eclampsia (the end-point) was defined as the time of the firstelevated blood-pressure or urine protein measurement leading to thediagnosis of pre-eclampsia. A small-for-gestational-age infant wasdefined as an infant whose birth weight was below the 10^(th) percentileaccording to U.S. tables of birth weight for gestational age thataccounted for race, parity, and infant gender.

Procedures

Assays were performed by personnel who were unaware of pregnancyoutcomes. Specimens were randomly ordered for analysis. Enzyme-linkedimmunosorbent assays (ELISAs) for sFlt, free PlGF, and free VEGF wereperformed in duplicate, as previously described, with the use ofcommercial kits (R&D Systems, Minn.). The minimal detectable doses inthe assays for sFlt1, PlGF, and VEGF were 5, 7, and 5 pg per milliliter,respectively, with inter-assay and intra-assay coefficients of variationof 7.6 and 3.3 percent, respectively, for sFlt1; 10.9 and 5.6 percent,for PlGF; and 7.3 and 5.4 percent, for VEGF. Urinary creatinine wasmeasured using a commercially available picric acid calorimetric assay(Metra creatinine assay kit, Quidel Corp., CA).

Statistical Analysis

The chi-square test was used for comparison of categorical variables;and the t-test, for comparison of continuous variables. Althougharithmetic mean concentrations are reported in the text and figures,statistical testing was conducted after logarithmic transformation,using the SAS/PROC GENMOD procedure (SAS v8.0, Cary, N.C.) in crude andadjusted analyses to account for subjects with varying numbers ofspecimens. Odds ratios were adjusted using logistic-regression analysis.

Results

Of the 120 women with pre-eclampsia, 80 had mild and 40 had severedisease. Compared with controls, women with pre-eclampsia had greaterbody-mass index, higher blood pressure at enrollment in the CPEP trial,and larger proportions of their current pregnancies complicated bypreterm delivery or resulting in small-for-gestational-age infants.Patient and infant characteristics have been described previously andare briefly summarized in Table 3.

Differences in Urinary PlGF after Onset of Pre-eclampsia

We first ascertained that urinary levels of PlGF were altered in womenafter development of clinical pre-eclampsia. Among 22 pairs of womenwith pre-eclampsia and gestational-age matched controls, end-pointspecimens had lower levels of PlGF than specimens from controls (meanPLGF level, 32 vs. 234 pg/ml, P<0.001; and 50 vs 227 pg per mgcreatinine, P<0.001).

Gestational Changes in Urinary PlGF

To evaluate gestational patterns, we performed cross-sectional analysesof urine obtained within gestational-age intervals of four to fiveweeks, with PlGF levels expressed as concentrations (FIG. 9A) or as pgper mg creatinine (FIG. 9B). The P values in FIG. 9A are for thecomparisons, after logarithmic transformation, with specimens fromcontrols obtained during the same gestational-age interval andaccounting for subjects with varying numbers of specimens. Thedifferences, after logarithmic transformation, between the specimensobtained at 29-36 weeks from women who already had clinicalpre-eclampsia and those obtained at 29-36 weeks from women in whompre-eclampsia later developed were also significant (P<0.001 for thecomparison at 29-32 weeks, P<0.001 for the comparison at 33-36 weeks,and P=0.003 for the comparison at 37-42 weeks). Note that PlGFconcentrations before onset of pre-eclampsia do not include endpointspecimens obtained after appearance of hypertension or proteinuria. FIG.9A also shows the mean serum concentrations of PlGF for the women whosubsequently develop pre-eclampsia after excluding specimens obtainedwithin 5 weeks before onset of pre-eclampsia (broken red line). Thegraph in FIG. 9B shows that the differences, after logarithmictransformation, between the specimens obtained at 29-36 weeks from womenwho already had clinical pre-eclampsia and those obtained at 29-36 weeksfrom women in whom pre-eclampsia later developed were also significant(P=0.004 for the comparison at 29-32 weeks, P<0.001 for the comparisonat 33-36 weeks, and P=0.02 for the comparison at 37-42 weeks).

The PlGF levels in controls increased during the first two trimesterswith a more rapid increase after 21-24 weeks, reaching a peak at 29-32weeks, and decreasing thereafter. The levels in women who subsequentlydeveloped pre-eclampsia followed a similar pattern, but weresignificantly lower at 25-28, 29-32, and 33-36 weeks. When specimensobtained within 5 weeks before the onset of pre-eclampsia were excluded,the differences in the preceding gestational age intervals between thecontrols and women who later had pre-eclampsia were less pronounced.Among women with specimens obtained in the same gestational-ageinterval, those who already had clinical pre-eclampsia had significantlylower concentrations at 29-32, 33-36, and 37-42 weeks than those whodeveloped pre-eclampsia later. Similar gestational age patterns amongcontrols and cases before and after onset of clinical pre-eclampsia wereobserved when restricting the analysis of specimens either to firstmorning (FIG. 9C) or random (FIG. 9D) urines.

Relationship of Urinary PlGF to Severity of Pre-eclampsia

Before the onset of pre-eclampsia, there were particularly largedifferences between the levels of urinary PlGF in controls and those inwomen who later had pre-eclampsia with onset before 37 weeks or who hadpre-eclampsia and a small-for-gestational-age infant. FIG. 10 shows PlGFconcentrations and PlGF expressed as pg per mg creatinine between 21-32weeks of gestation.

Alterations in urinary PlGF levels were also more pronounced in womenwho subsequently developed pre-eclampsia before term (<37 weeks ofgestation) than in women who had an onset of pre-eclampsia at term (≧37weeks) (at 21-32 weeks: PlGF concentration, 87 pg/ml in women withpre-eclampsia before term vs. 223 pg/ml in women with pre-eclampsia atterm, P<0.001; at 33-42 weeks: PlGF concentration, 22 pg/ml in womenwith pre-eclampsia before term vs. 118 pg/ml in women with pre-eclampsiaat term, P<0.001). Results were similar when using PlGF expressed as pgper mg creatinine or after adjusting PlGF concentrations for creatinine,gestational age at specimen collection, storage time, body mass index,and maternal age. Furthermore, PlGF levels in specimens obtained beforeonset of pre-eclampsia from women who later had pre-eclampsia and asmall-for-gestational-age infant were lower than in women who later hadpre-eclampsia, but whose infants were not small-for-gestational-age (at21-32 weeks: PlGF concentration, 62 vs. 205 pg/ml, P=0.002; at 33-42weeks: PlGF concentration, 42 vs. 123 pg/ml, P=0.06).

Odds Ratios for Pre-eclampsia Associated with Urinary PlGF

To determine the risk of pre-eclampsia according to urinary PlGF inspecimens obtained before the onset of clinical signs, we divided PlGFvalues into quartiles based on the distribution in controls andcalculated adjusted odds ratios for pre-eclampsia in each quartile, ascompared to the highest quartile (Table 6) or to all other quartiles(described below).

TABLE 6 Odds Ratios for Pre-eclampsia at Less Than 37 Weeks of Gestationand at 37 Weeks or More of Gestation According to Quartile* of UrinaryPIGF No. of Control PE < 37 wk PE ≦ 37 wks PIGF Specimens No. spec. OR(95% C.I.)** No. spec. OR (95% C.I.)** 13-20 wks Q1: ≦29 pg/ml 25 6  0.6(0.2-2.4) 19 0.9 (0.3-2.3) Q2: 29-59 pg/ml 24 12  1.3 (0.4-4.3) 25 1.4(0.6-3.3) Q3: 59-88 pg/ml 24 5  0.7 (0.2-2.7) 19 1.1 (0.5-2.8) Q4: >88pg/ml 24 6  1.0 17 1.0 21-32 wks Q1: ≦118 pg/ml 29 30 31.3 (5.6-174.7)33 2.2 (1.0-5.1) Q2: 118-230 pg/ml 29 4  2.6 (0.4-16.8) 21 1.3 (0.6-3.0)Q3: 230-309 pg/ml 29 1  0.6 (0.1-7.6) 11 0.7 (0.3-1.7) Q4: >309 pg/ml 292  1.0 18 1.0 33-41 wks Q1: ≦55 pg/ml 25 2 N/A 31 4.2 (1.4-12.5) Q2:55-113 pg/ml 25 0 N/A 21 2.5 (0.8-7.7) Q3: 113-318 pg/ml 25 0 N/A 17 2.1(0.7-6.5) Q4: >318 pg/ml 24 0 N/A 6 1.0 pg PIGF/mg Creatinine 13-20 wksQ1: ≦26 pg/mg 25 8  0.5 (0.1-2.2) 21 0.9 (0.3-2.5) Q2: 26-52 pg/mg 24 9 0.7 (0.2-3.0) 25 1.3 (0.5-3.2) Q3: 52-78 pg/mg 24 5  0.4 (0.1-1.8) 150.8 (0.3-2.2) Q4: >78 pg/mg 24 7  1.0 19 1.0 21-32 wks Q1: ≦120 pg/mg 2929 15.4 (3.7-64.3) 33 2.6 (1.1-6.3) Q2: 120-180 pg/mg 29 2  0.9(0.1-6.1) 13 1.0 (0.4-2.6) Q3: 180-323 pg/mg 29 3  0.9 (0.2-5.1) 22 1.7(0.7-4.0) Q4: >323 pg/mg 29 3  1.0 15 1.0 33-41 wks Q1: ≦69 pg/mg 24 2N/A 34 2.6 (1.0-6.6) Q2: 69-153 pg/mg 25 0 N/A 23 1.7 (0.6-4.5) Q3:153-268 pg/mg 25 0 N/A 8 0.6 (0.2-1.8) Q4: >268 pg/mg 24 0 N/A 10 1.0Quartiles were determined on the basis of control specimens Odds ratioswere adjusted for gestational age at specimen collection, specimenstorage time, maternal age and body mass index (with 95% ConfidenceIntervals). The reference category was the highest quartile: Q4.Specimens from cases were all obtained before onset of clinical signs ofpre-eclampsia.

Among specimens obtained at 21-32 weeks of gestation the lowest quartileof PlGF was associated with a greatly increased risk of pretermpre-eclampsia and a small increased risk of pre-eclampsia at term. Forpreterm pre-eclampsia, after adjustment for gestational age at specimencollection, storage time, body mass index, and age, using PlGFconcentration the odds ratio for the lowest quartile vs. all others was22.5, 95% confidence interval, 7.4-67.8; and using pg PlGF per mgcreatinine the odds ratio was 16.4, 95% confidence interval, 5.9-45.5.After restricting specimens to first morning urines, adjusted oddsratios were 39.5 with 95% confidence interval, 6.5-240.8; and 20.4 with95% confidence interval, 4.5-92.3, for PlGF concentration and PlGF permg creatinine, respectively. Using random urine specimens, adjusted oddsratios were 13.5 with 95% confidence interval, 2.3-79.8; and 11.1 with95% confidence interval, 2.0-61.3, respectively. For term pre-eclampsia,after adjustment for the factors noted above and using all urinespecimens, odds ratios were 2.2 with 95% confidence interval, 1.2-4.3;and 2.1, 95% confidence interval, 1.1-4.1, respectively. For specimensobtained at 13 to 20 weeks of gestation, the lowest quartile of PlGF wasneither associated with an increased risk of preterm, nor of termpre-eclampsia. However, the lowest quartile of PlGF was associated withan increased risk of term pre-eclampsia vs. all other quartiles inspecimens obtained at 33-42 weeks of gestation: adjusted odds ratio 2.3with 95% confidence interval, 1.2-4.5, for pg PlGF per mg creatinine.

When we performed the same analyses in specimens obtained at 21-32 weeksof gestation for women who developed pre-eclampsia complicated by asmall-for-gestational-age infant, we found that the estimates wereunstable (adjusted OR 405, 95% confidence interval, 27-5983, for pg PlGFper mg creatinine). This was because there were only 20 such women, allof whom were in the lowest (N=19) or next lowest (N=1) quartiles ofurinary PlGF. Nevertheless, the data indicate that low urinary PlGF isassociated with a substantial increase in risk for pre-eclampsia with asmall-for-gestational-age infant.

Gestational Changes in Urinary PlGF within Individual Women

FIG. 11 depicts longitudinally the changes in PlGF concentration within13 patients with preterm pre-eclampsia (pre-eclampsia <37 weeks) and 13controls with gestational-age matched specimens. All 13 women whodeveloped pre-eclampsia before 37 weeks of gestation were selected whohad at least a baseline urine, a urine obtained within 21-32 weeks ofgestation, and an end-point urine, which might also serve as the 21-32week specimen. Each case was matched to a control with the same orgreater number of specimens obtained at similar gestational ages. Onecontrol had very low urinary PlGF per mg creatinine throughoutpregnancy: 17, 5, and 0 pg/ml at 116, 177, and 248 days of gestation,respectively. This woman had a single episode of 1+proteinuria and asingle diastolic blood pressure of 90 mm Hg recorded 2 hours beforedelivery on day 266 of gestation. Patients with preterm pre-eclampsiahad lower levels of PlGF usually throughout gestation, whereas controlstended to have levels which increased with advancing gestation and fellnear term.

Relationship of Urinary PlGF to Proximity to Pre-eclampsia

Urinary concentrations of PlGF in specimens obtained at 21-32 weeks ofgestation and within five weeks before the onset of pre-eclampsia werelower (43 pg/ml) than in specimens obtained more than five weeks beforeclinical disease (196 pg/ml, P<0.001). In specimens obtained at 33-42weeks of gestation concentrations were 110 pg/ml vs. 187 pg/ml,respectively (P=0.05). There was little difference when PlGF wasnormalized for creatinine.

FIG. 12A is a scatter plot of urinary PlGF concentrations at 21-32 weeksfrom 69 controls and 20 cases who subsequently developed pre-eclampsiabefore term (<37 weeks). Women who developed pre-eclampsia before termhad lower urinary PlGF concentrations than normotensive controls.Concentrations were lowest (i.e., less than 150 pg/ml) in specimensobtained within five weeks before the onset of clinical disease.However, a number of control specimens also had low urinary PlGF. Inorder to distinguish these specimens from specimens obtained within fiveweeks prior to pre-eclampsia, we examined serum measurements of theratio of sFlt1 to PlGF. The ratio accounts for both the increased sFlt1and decreased PlGF observed before onset of pre-eclampsia. A scatterplot of the ratios of sFlt1 to PlGF concentrations in paired sera isgiven in FIG. 12B. Ratios are elevated (>5) in all specimens obtainedwithin five weeks before the onset of pre-eclampsia and exceed almostall control values.

Urinary sFlt1 and Urinary VEGF in Pre-Eclampsia

We randomly selected 22 cases and 22 controls for analysis of urinarysFlt1 and VEGF within 21-32 weeks of gestation before onset of clinicalpre-eclampsia. In 16 of 22 case specimens (73%) and 19 of 22 controlspecimens (86%) urinary sFlt1 was undetectable. In contrast, urinaryVEGF was detected in all specimens, but was not significantly altered incases before or after the onset of hypertension and proteinuria (before:272 vs. 248 pg/ml in the groups of 22 randomly selected cases andcontrols, respectively, P=0.56; after: 167 vs. 103 pg/ml in 22gestational-age matched cases and controls, respectively, P=0.61).

Conclusions

In this study of 120 women with pre-eclampsia and 118 normotensivecontrols, urinary concentrations of PlGF were significantly lowerbeginning at 25-28 weeks of gestation among the women who subsequentlydeveloped pre-eclampsia. Differences between the two groups became morepronounced at 29-36 weeks. We have previously shown that serum free PlGFwas lower in cases than controls beginning at 13-16 weeks of gestation,becoming even lower after 25 weeks of gestation. As with serummeasurements, in the current study urinary PlGF at 21-32 weeks ofgestation was significantly decreased in those who developedpre-eclampsia before 37 weeks or complicated by asmall-for-gestational-age infant and within 5 weeks of the onset ofclinical signs. Furthermore, among women in the lowest quartile ofurinary PlGF concentrations (<118 pg/ml) at 21-32 weeks of gestation,the risk of developing pre-eclampsia before 37 weeks of gestation orcomplicated by a small-for-gestational-age infant was markedly elevated.The risk was high, irrespective of adjustment for urinary creatinineconcentrations, and evident even in random urines. The association was,however, stronger with first morning specimens, which are likely to bemore concentrated. Thus, urinary PlGF was especially useful foridentifying the patients who would benefit most from early diagnosis. Wehave also demonstrated that a strategy of following urine measurement ofPlGF with serum measurements of sFlt1 and PlGF in selected patients mayminimize false positives from urine testing.

Urinary VEGF concentrations were reported recently to be modestlyelevated in 37 women with severe pre-eclampsia, compared to 32 withuncomplicated pregnancy. We found non-significant elevations of urinaryVEGF before and after the onset of pre-eclampsia, consistent with ourhypothesis that urinary VEGF reflects primarily local renal VEGFproduction. Since urinary VEGF originates almost entirely from renalpodocyte and tubular cells, it has not been exposed to circulatingsFlt1, which is too large a molecule to filter freely through an intactglomerulus. Therefore, while reduced urinary PlGF in women withpre-eclampsia likely reflects reduced circulating free PlGF, the resultof binding to excess circulating sFlt1, levels of urinary VEGF do notreflect the angiogenic imbalance in the blood.

The identification of angiogenic proteins which appear to mediate thematernal syndrome of pre-eclampsia may present specific targets fortherapeutic intervention to restore the appropriate angiogenic balance(Maynard et al., supra). Prevention and treatment are especially neededfor women with early onset pre-eclampsia or pre-eclampsia complicated bya small-for-gestational-age infant. However, such women must first beidentified before the onset of clinical disease. These data demonstratethat a reliable and valid dipstick assay can be developed and used toscreen all women for low urinary PlGF concentrations. As a follow-up forthose women identified with low levels of urinary PlGF, serial serummeasurements of sFlt1 and PlGF could then be used to identify moreprecisely individuals at high risk.

EXAMPLE 12 Ancillary Study Demonstrating Urinary PlGF DuringMid-Pregnancy is a Specific Predictor of Pre-Eclampsia

We performed an ancillary study to ascertain whether urinary PlGF at21-32 weeks of gestation might differ between women with male or femaleinfants and to determine if concentrations of urinary PlGF might belower than normal in women with gestational hypertension and in womenwho remained normotensive during pregnancy, but delivered asmall-for-gestational-age (SGA) infant. Among the 4256 women in the CPEPtrial with adequate data who delivered a liveborn infant not known tohave a chromosomal abnormality, we excluded 239 with term preeclampsia(≧37 weeks). Of the 4017 women remaining, 3303 had at least one urinespecimen obtained within 21-32 weeks of gestation before onset of laboror delivery and before onset of preeclampsia or gestationalhypertension. Among these women we randomly selected 120 whose pregnancywas normotensive and whose infant was not SGA, 60 with normotensivepregnancy who delivered an SGA infant, 60 with gestational hypertension,and 59 with preterm (<37 weeks) preeclampsia. In each group we chosehalf the women to have delivered male infants and half, female infants,except for the group with preterm preeclampsia. In this group weselected 30 with male infants, but could find only 29 with femaleinfants. PlGF was analyzed in all urine specimens obtained at 21-32weeks gestation.

Preeclampsia, Gestational Hypertension, Small-for-Gestational-Age, andInstitutional Review Board

Preeclampsia was defined as a newly elevated diastolic blood pressure ofat least 90 mm Hg and proteinuria of at least 1+ (30 mg per deciliter)on dipstick testing, each on two occasions 4 to 168 hours apart. Severepreeclampsia was defined as the HELLP syndrome (hemolysis, elevatedliver-enzyme levels, and a low platelet count), eclampsia, orpreeclampsia with either severe hypertension (diastolic bloodpressure >110 mm Hg) or severe proteinuria (urinary protein excretion≧3.5 g per 24 hours or findings of ≧3+[300 mg per deciliter] on dipsticktesting). Gestational hypertension was hypertension as defined above inthe absence of proteinuria. Detailed definitions have been published(Levine et al., N. Engl. J. Med. 337:69-76 (1997) and Levine et al.,Control Clin. Trials 17:442-469 (1996)). The time of onset ofpreeclampsia was defined as the time of the first elevatedblood-pressure or urine protein measurement leading to the diagnosis ofpreeclampsia. Similarly the onset of gestational hypertension was thetime of the first elevated blood-pressure which led to the diagnosis. Asmall-for-gestational-age infant was an infant whose birth weight wasbelow the 10^(th) percentile according to U.S. tables of birth weightfor gestational age that accounted for race, parity, and infant gender(Zhang et al., Obstet. Gynecol. 86:200-208 (1995)). Because the studyused data and specimens that could not be linked to identifiable women,the office of Human Subjects Research of the National Institutes ofHealth granted it an exemption from the requirement for review andapproval by the institutional review board.

Procedures

Assays were performed by personnel who were unaware of pregnancyoutcomes. Specimens were randomly ordered for analysis. Enzyme-linkedimmunosorbent assays (ELISAs) for sFlt1, free PlGF, and free VEGF wereperformed in duplicate, as previously described, with the use ofcommercial kits (R&D Systems, MN). (Maynard et al., supra) The minimaldetectable doses in the assays for sFlt1, PlGF, and VEGF were 5, 7, and5 pg per milliliter, respectively, with inter-assay and intra-assaycoefficients of variation of 7.6 and 3.3 percent, respectively, forsFlt1; 10.9 and 5.6 percent, for PlGF; and 7.3 and 5.4 percent, forVEGF. The ELISA kits for sFlt1, VEGF and PlGF were validated for use inurine specimens with 96%, 98% and 99% recovery from spiked urinesamples, respectively. Urinary creatinine was measured using acommercially available picric acid colorimetric assay (Metra creatinineassay kit, Quidel Corp., CA).

Statistical Analysis

The chi-square test was used for comparison of categorical variables;and the t-test, for comparison of continuous variables. Althougharithmetic mean concentrations are reported in the text and figures,statistical testing was conducted within each time interval individuallyafter logarithmic transformation, using the generalized estimatingequations (GEE) method (SAS/PROC GENMOD procedure, SAS v8.0, Cary, N.C.)in crude and adjusted analyses to account for subjects with varyingnumbers of specimens. Odds ratios were adjusted with the use oflogistic-regression analysis. Since matching was complete only foranalyses of the earliest serum specimen in the entire study population,matching was not accounted for in the statistical analyses.

Results

In order to test further the hypothesis that decreased urinary PlGF isspecific for early onset preeclampsia, we performed a second study inwhich we analyzed urine specimens obtained at 21-32 weeks from womenwith other obstetrical conditions which may share similarities ofpathogenesis. We compared women with gestational hypertension and womenwho remained normotensive during pregnancy, but delivered an SGA infant,to normotensive women whose infant was not SGA (controls) and to womenwith preeclampsia before 37 weeks. The clinical characteristics of thewomen in this study and of their newborn infants are summarized in Table7. The characteristics of women with preeclampsia and their infants weresimilar to those reported for such women in the main study.

TABLE 7 Characteristics of women in the ancillary study at CPEPenrollment and of their newborn infants Normotensive NormotensiveGestational Without SGA With SGA Hypertension PE < 37 Wks Characteristic(n = 120) (n = 60) (n = 60) (n = 59) Age (yr)  21.8 ± 4.6  21.3 ± 4.9 22.2 ± 5.3  21.1 ± 4.7 Body mass index  25.8 ± 6.1  22.8 ± 3.6***  28.3± 7.4*  27.6 ± 6.9 Systolic blood pressure (mm Hg)  106 ± 9  106 ± 8 108 ± 9  111 ± 8† Diastolic blood pressure (mm Hg)  60 ± 7  60 ± 8  62± 9  65 ± 7*** Gestational age at delivery (wks)  39.0 ± 1.8  38.7 ± 1.4 39.6 ± 1.7  34.6 ± 2.3*** Current smoker [n (%)] 15 (12.5) 13 (21.7)  3(5.0)  4 (6.8) Ever married [n (%)] 34 (28.3) 16 (26.7) 15 (25.4) 16(27.1) Race/ethnicity^(φ) White, non-Hispanic [n (%)] 46 (38.3) 20(33.3) 20 (33.3) 16 (27.1) White, Hispanic [n (%)] 16 (13.3) 16 (26.7) 6 (10.0) 10 (17.0) African-American [n (%)] 55 (45.8) 24 (40.0) 33(55.0) 30 (50.9) Other, unknown [n (%)]  3 (2.5)  0 (0.0)  1 (1.7)  3(5.1) Birthweight (g) 3273 ± 456 2538 ± 278*** 3437 ± 559** 2193 ±726*** Delivery <37 wks [n (%)] 13 (10.8)  6 (10.0)  3 (5.0) 50(84.8)*** Small for gestational age  0 (0.0) 60 (100.0)***  2 (3.3) 18(30.5)*** (<10^(th) percentile) [n (%)] Mean ± standard deviation unlessindicated P-values for the difference with “Normotensive without SGA” *P= 0.02 **P = 0.04 ***P < 0.001 †P = 0.001 ^(φ)Race or ethnicity wasself-reported.

Compared to normotensive women whose infants were not SGA, women withgestational hypertension had greater body-mass index and infants ofgreater birthweight; and normotensive women with an SGA infant, lowerbody-mass index and infants of lower birthweight. Normotensive womenwith SGA infants were most likely and women with hypertensive disordersof pregnancy, least likely to have smoked during pregnancy.

FIG. 13 depicts urinary PlGF at 21-32 weeks of gestation expressed asconcentrations (pg/ml) and as pg per mg creatinine. PlGF levels in womenwho remained normotensive during pregnancy, but delivered an SGA infant,did not differ from those of normotensive controls whose infant was notborn SGA. Similarly, levels in subjects with gestational hypertensiondid not differ from those of normotensive controls. However, levels ofurinary PlGF in patients who developed preeclampsia before 37 weeks ofgestation—collected on average 42 days prior to clinical disease—weremuch lower than controls (77 vs. 206 pg/ml, p<0.0001). Within each groupPlGF concentrations among women who delivered male or female infants didnot differ significantly.

Conclusions

Urinary PlGF was much lower at 21-32 weeks of gestation in women whodeveloped preeclampsia before 37 weeks than in women who developedgestational hypertension or delivered a small-for-gestational-ageinfant, two obstetrical conditions with similarities to preeclampsia.Thus, a low urinary PlGF concentration at this stage of pregnancy canlikely distinguish preeclampsia from gestational hypertension andintrauterine growth retardation.

Diagnostics

The present invention features diagnostic assays for the detection ofpre-eclampsia, eclampsia, or the propensity to develop such conditions.Levels of VEGF, PlGF, or sFlt-1, either free or total levels, aremeasured in a subject sample and used as an indicator of pre-eclampsia,eclampsia, or the propensity to develop such conditions.

In one embodiment, a metric is used to determine whether a relationshipbetween levels of at least two of the proteins is indicative ofpre-eclampsia or eclampsia. Standard methods may be used to measurelevels of VEGF, PlGF, or sFlt-1 polypeptide in any bodily fluid,including, but not limited to, urine, serum, plasma, saliva, amnioticfluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA,“sandwich assays”, western blotting using antibodies directed to VEGF,PlGF or sFlt-1, immunodiffusion assays, agglutination assays,fluorescent immunoassays, protein A or G immunoassays, andimmunoelectrophoresis assays and quantitative enzyme immunoassaytechniques such as those described in Ong et al. (Obstet. Gynecol.98:608-611, 2001) and Su et al. (Obstet. Gynecol., 97:898-904, 2001).ELISA assays are the preferred method for measuring levels of VEGF,PlGF, or sFlt-1. Particularly preferred, for ease and simplicity ofdetection, and its quantitative nature, is the sandwich or doubleantibody assay of which a number of variations exist, all of which arecontemplated by the present invention. For example, in a typicalsandwich assay, unlabeled antibody that recognizes the antigen (i.e.,sFlt-1, PlGF, or VEGF polypeptide) is immobilized on a solid phase, e.g.microtiter plate, and the sample to be tested is added. After a certainperiod of incubation to allow formation of an antibody-antigen complex,a second antibody, labeled with a reporter molecule capable of inducinga detectable signal, is added and incubation is continued to allowsufficient time for binding with the antigen at a different site,resulting with a formation of a complex of antibody-antigen-labeledantibody. The presence of the antigen is determined by observation of asignal which may be quantitated by comparison with control samplescontaining known amounts of antigen.

Elevated serum levels of sFlt-1 are considered a positive indicator ofpre-eclampsia. This value of sFlt-1 may be preferentially 2 ng/ml ormore. Additionally, any detectable alteration in levels of sFlt-1, VEGF,or PlGF relative to normal levels is indicative of eclampsia,pre-eclampsia, or the propensity to develop such conditions. Preferably,sFlt-1 is measured, more preferably measurement of VEGF and PlGF arecombined with this measurement, and most preferably all three proteins(or mRNA levels indicative of protein levels) are measured. Inadditional preferred embodiments, the body mass index (BMI) andgestational age of the fetus is also measured and included thediagnostic metric.

In another embodiment, the PAAI (sFlt-1/VEGF+PlGF) is used as ananti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia, orthe propensity to develop such conditions. If the PAAI is greater than10, more preferably greater than 20, then the subject is considered tohave pre-eclampsia, eclampsia, or to be in imminent risk of developingthe same. The PAAI (sFlt-1/VEGF+PlGF) ratio is merely one example of auseful metric that may be used as a diagnostic indicator. It is notintended to limit the invention. Virtually any metric that detects analteration in the levels of any of sFlt-1, PlGF, or VEGF in a subjectrelative to a normal control may be used as a diagnostic indicator.

Expression levels of particular nucleic acids or polypeptides may becorrelated with a particular disease state (e.g., pre-eclampsia oreclampsia), and thus are useful in diagnosis. Oligonucleotides or longerfragments derived from a sFlt-1, PlGF, or VEGF nucleic acid sequence maybe used as a probe not only to monitor expression, but also to identifysubjects having a genetic variation, mutation, or polymorphism in ansFlt-1, PlGF, or VEGF nucleic acid molecule that are indicative of apredisposition to develop the conditions. Such polymorphisms are knownto the skilled artisan and are described, for example, by Parry et al.(Eur. J. Immunogenet. 26:321-3, 1999). A survey of the GenBank database(www.ncbi.nlm.nih.gov) reveals at least 330 known polymorphisms in thegene and the promoter region of Flt-1/sFlt-1. These polymorphisms mayaffect sFlt-1 nucleic acid or polypeptide expression levels orbiological activity. Detection of genetic variation, mutation, orpolymorphism relative to a normal, reference sample can be as adiagnostic indicator of pre-eclampsia, eclampsia, or the propensity todevelop pre-eclampsia or eclampsia.

Such genetic alterations may be present in the promoter sequence, anopen reading frame, intronic sequence, or untranslated 3′ region of ansFlt-1 gene. Information related to genetic alterations can be used todiagnose a subject as having pre-eclampsia, eclampsia, or a propensityto develop such conditions. As noted throughout, specific alterations inthe levels of biological activity of sFlt-1, VEGF, and/or PlGF can becorrelated with the likelihood of pre-eclampsia or eclampsia, or thepredisposition to the same. As a result, one skilled in the art, havingdetected a given mutation, can then assay one or more metrics of thebiological activity of the protein to determine if the mutation causesor increases the likelihood of pre-eclampsia or eclampsia.

In one embodiment, a subject having pre-eclampsia, eclampsia, or apropensity to develop such conditions will show an increase in theexpression of a nucleic acid encoding sFlt-1 or an alteration in PlGF orVEGF levels. Methods for detecting such alterations are standard in theart and are described in Ausubel et al., supra. In one example northernblotting or real-time PCR is used to detect sFlt-1, PlGF, or VEGF mRNAlevels.

In another embodiment, hybridization with PCR probes that are capable ofdetecting an sFlt-1 nucleic acid molecule, including genomic sequences,or closely related molecules, may be used to hybridize to a nucleic acidsequence derived from a subject having pre-eclampsia or eclampsia or atrisk of developing such conditions. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), determine whether the probehybridizes to a naturally occurring sequence, allelic variants, or otherrelated sequences. Hybridization techniques may be used to identifymutations indicative of a pre-eclampsia or eclampsia in an sFlt-1nucleic acid molecule, or may be used to monitor expression levels of agene encoding an sFlt-1 polypeptide (for example, by Northern analysis,Ausubel et al., supra).

In yet another embodiment, humans may be diagnosed for a propensity todevelop pre-eclampsia or eclampsia by direct analysis of the sequence ofan sFlt-1, VEGF, or PlGF nucleic acid molecule.

A subject having pre-eclampsia, eclampsia, or a propensity to developsuch conditions will show an increase in the expression of an sFlt-1polypeptide. The sFlt-1 polypeptide can include full-length sFlt-1,degradation products, alternatively spliced isoforms of sFlt-1,enzymatic cleavage products of sFlt-1, and the like. An antibody thatspecifically binds an sFlt-1 polypeptide may be used for the diagnosisof pre-eclampsia or eclampsia or to identify a subject at risk ofdeveloping such conditions. A variety of protocols for measuring analteration in the expression of such polypeptides are known, includingimmunological methods (such as ELISAs and RIAs), and provide a basis fordiagnosing pre-eclampsia or eclampsia or a risk of developing suchconditions. Again, an increase in the level of the sFlt-1 polypeptide isdiagnostic of a subject having pre-eclampsia, eclampsia, or a propensityto develop such conditions.

In one embodiment, the level of sFlt-1, VEGF, or PlGF polypeptide ornucleic acid, or any combination thereof, is measured at least twodifferent times and an alteration in the levels as compared to normalreference levels over time is used as an indicator of pre-eclampsia,eclampsia, or the propensity to develop such conditions. In anotherembodiment, the level of sFlt-1, VEGF, or PlGF polypeptide or nucleicacid, or any combination thereof is compared to the level in a referencesample.

The level of sFlt-1, VEGF, or PlGF polypeptide can also be compared to astandard curve to determine if it falls within “normal ranges” of thelevel of polypeptide. In this embodiment, a standard curve isestablished for each of the polypeptides using purified or recombinantforms (e.g., greater than 80%, 90%, 95%, 99% or 100% pure) of thepolypeptide for comparison. A standard curve is generated and theconcentration of the polypeptide is determined by comparison to astandard curve established for the same polypeptide. For example, astandard curve can be established for sFlt-1 and a subject sample that,when compared to the standard curve, has sFlt-1 concentrations greaterthan 2 ng/mL is considered indicative of pre-eclampsia, eclampsia, orthe propensity to develop such conditions.

The level of sFlt-1, VEGF, or PlGF in the bodily fluids of a subjecthaving pre-eclampsia, eclampsia, or the propensity to develop suchconditions may be altered (increased or decreased) by as little as 10%,20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or morerelative to the level of sFlt-1, VEGF, or PlGF in a normal control. Thelevel of sFlt-1 present in the bodily fluids of a subject havingpre-eclampsia, eclampsia, or the propensity to develop such conditionsmay be increased by 1.5-fold, 2-fold, 3-fold, 4-fold or even by as muchas 10-fold or more relative to levels in a normal control subject.

In one embodiment, a subject sample of a bodily fluid (e.g., urine,plasma, serum, amniotic fluid, or cerebrospinal fluid) is collectedearly in pregnancy prior to the onset of pre-eclampsia symptoms. Inanother example, the sample can be a tissue or cell collected early inpregnancy prior to the onset of pre-eclampsia symptoms. Non-limitingexamples include placental tissue, placental cells, endothelial cells,and leukocytes such as monocytes. In humans, for example, maternal bloodserum samples are collected from the antecubital vein of pregnant womenduring the first, second, or third trimesters of the pregnancy.Preferably, the assay is carried out during the first trimester, forexample, at 4, 6, 8, 10, or 12 weeks, or during the second trimester,for example at 14, 16, 18, 20, 22, or 24 weeks. Such assays may also beconducted at the end of the second trimester or the third trimester, forexample at 26, 28, 30, 32, 34, 36, 37, 38, 39, or 40 weeks. It ispreferable that levels of sFlt-1, VEGF, or PlGF be measured twice duringthis period of time. For the diagnosis of post-partum pre-eclampsia oreclampsia, assays for sFlt-1, VEGF, or PlGF may be carried outpostpartum.

In one particular example, serial blood samples can be collected duringpregnancy and the levels of soluble sFlt-1 determined by ELISA. In onestudy using this technique, the alternatively spliced mRNA encodingsFlt-1 is highly expressed by trophoblast cells and the protein wasreadily detectable in the plasma of pregnant women. It was observed thatthe levels of sFlt-1 increased approximately 3-fold between 20 and 36weeks gestation. Levels were observed to be significantly higher inhigh-risk women who subsequently went on to develop pre-eclampsia(Charnock-Jones et al., J. Soc. Gynecol. Investig. 10(2):230, 2003).

In one preferred embodiment, PlGF polypeptide levels are measured in abodily fluid sample, preferably urine, and used as a diagnosticindicator of pre-eclampsia, eclampsia, or the propensity to develop thesame. Measurements of PlGF polypeptide levels in the urine can also beused as an initial assessment of the potential risk for pre-eclampsia oreclampsia and a woman determined to be “at risk” by PlGF measurementscan then undergo additional diagnostic assays such as the ones describedherein or known in the art. In one example, a woman diagnosed with arisk of developing pre-eclampsia or eclampsia by PlGF polypeptidemeasurement in a urine sample is further monitored by serum analysis ofVEGF, sFlt-1, and/or PlGF levels as described above. In another example,the PAAI is determined using the serum values for each of thesepolypeptides. A woman identified as having a risk of developingpre-eclampsia or eclampsia by urine analysis for PlGF can be monitoredregularly prior to pregnancy, throughout the pregnancy (e.g., everymonth, every three weeks, every two weeks, weekly, every third day,every other day, or daily), or after the pregnancy.

The free form of PlGF has an average molecular weight of about 30 kDaand is small enough to be filtered by the kidney and released into theurine. PlGF, when complexed to sFlt-1, has a much greater molecularweight and would therefore not be released into the urine. Although notwishing to be bound by theory, the inventors have discovered that duringpre-eclampsia, when the levels of sFlt-1 are increased, sFlt-1 cancomplex to PlGF, thereby reducing the levels of free PlGF released intothe urine. As a result, urine analysis for free PIGF levels can be usedto diagnose pre-eclampsia or eclampsia or a patient at risk for havingthe same. In order to detect free PlGF, it is preferred that an antibodythat specifically recognizes free PlGF is used for these assays. Such anantibody can recognize, for example, the sFlt-1 binding domain of PlGF.Examples of such a specific antibody include the capture antibody usedin the human PlGF ELISA kit (catalog #DPG000, R&D Systems, Minneapolis,Minn.), monoclonal anti-placental growth factor (clone 37203.111,Sigma-Aldrich, St. Louis, Mo.). These antibodies recognize specificsequences in the N-terminal region of human PlGF protein. The sFlt1binding region to PlGF is between amino acids 39-105 of the PlGFprotein, wherein the total length of PlGF varies from 149 to 221 aminoacids depending on the isoform of PlGF. Additional preferred antibodiesinclude any antibody that recognizes the N-terminal region (preferablybetween amino acids 39-105 of PlGF) and that will specifically bind tofree PIGE and not PlGF bound to sFlt-1. Antibodies raised to C-terminuswill not have this property.

As with any of the diagnostic assays of the invention, PlGF levels in asubject sample can be compared to a reference sample to determinerelative levels A reference sample can be a urine sample from a patienthaving pre-eclampsia (generally having a level of free PlGF less than400 pg/ml, preferably less than 200 pg/ml) or from a normal urine sample(having a PlGF concentration ranging from 200 pg/ml to 800 pg/ml)depending on the desired use of the diagnostic assay. The PlGF levelscan also be compared to a reference value or standard to determineabsolute levels. The reference value or standard can be determined usinga standard curve established based on purified or recombinant forms(e.g., greater than 80%, 90%, 95%, 99% or 100% pure) of PlGF forcomparison. A value of PlGF less than 400 pg/ml, preferably less than200 pg/ml, and most preferably less than 100 pg/ml or a PlGF/creatininevalue less than 200 pg/mg of creatinine and preferably less than 100pg/mg of creatinine is considered a diagnostic indicator ofpre-eclampsia or eclampsia or a patient at risk for having the same. Forstandard curves, recombinant PlGF ranging from 10 pg/ml to 1 ng/ml canbe used. Other examples of recombinant proteins that can be used togenerate the standard curves include specific peptides that encompassthe amino terminus of PlGF, preferably amino acids 39-105 of the PlGFprotein (the region of PlGF that binds to sFlt-1). Alternatively, arecombinant PlGF/VEGF heterodimer (available commercially as catalog #297-VP, R &D Systems, MN) can also be used. The latter has the advantagethat this protein may also be used to generate the VEGF standard curvein the measurement of free VEGF.

ELISA assays are the preferred method for measuring levels of free PlGF.Particularly preferred, for ease and simplicity of detection, and itsquantitative nature, is the sandwich or double antibody ELISA assay ofwhich a number of variations exist, all of which are contemplated by thepresent invention. For example, in a typical sandwich assay, unlabeledantibody that recognizes the PlGF polypeptide is immobilized on a solidphase, e.g. microtiter plate, and the sample to be tested is added.After a certain period of incubation to allow formation of anantibody-antigen complex, a second antibody, labeled with a reportermolecule capable of inducing a detectable signal, is added andincubation is continued to allow sufficient time for binding with theantigen at a different site, resulting with a formation of a complex ofantibody-antigen-labeled antibody. The presence of the antigen isdetermined by observation of a signal which may be quantitated bycomparison with control samples containing known amounts of antigen.

In an example of the quantitative sandwich ELISA, a solid support (e.g.,a microtiter plate or a membrane) is pre-coated with an anti-PlGFbinding agent (e.g., a primary antibody). Standards or samples are addedto the substrate and PlGF, if present, will bind to the antibody. Astandardized preparation of enzyme-conjugated antibody that alsorecognizes PlGF is then added to “sandwich” the PlGF now immobilized onthe plate. The substrate is added and the enzyme and substrate areallowed to react over a short incubation period. The enzyme-substratereaction is terminated and the change is measured by art known methods(e.g., by eye, using a spectrophotometer, or measuringchemiluminescence). Such an assay can be used to determine the relativelevel of PlGF (e.g., as compared to the level in a reference sample,standard or level) or to determine the absolute concentration of PlGF.If so desired, the concentration of PlGF can be determined using a setof calibration standards of purified PlGF at varying concentrations. Thecalibration standards are assayed at the same time as the sample and areused to produce a standard curve measured by, for example, opticaldensity, versus PlGF concentration. The concentration of PlGF in thesample is then determined by comparing, for example, the optical densityof the samples to the standard curve. The concentrations of PlGF duringnormal pregnancy during mid-gestation and late-gestation will range from200-800 pg/ml depending on the gestational age of the mother. Any valueof urinary PlGF less than 400 pg/ml, preferably less than 200 pg/ml or avalue of urinary PlGF less than 200 pg/mg of creatinine will bediagnostic of preeclampsia. In general, the standard curves on the ELISAkit will include recombinant or purified PlGF at concentrations rangingfrom 10 pg/ml-1 ng/ml of PlGF.

In another example, an assay for detecting PlGF in a urine sampleincludes a membrane having an immobilized PlGF binding agent that isdetectably labeled in a manner that can distinguish between the PlGFwhen it is bound to free PlGF and when it is not bound to free PlGF.Preferred labels include fluorescent labels. The membrane is exposed tothe sample for a time sufficient to allow binding of the PlGF bindingagent to free PlGF present in the sample. The labeled PlGF binding agentbound to the free PlGF is then measured. Such an assay can be used todetermine the relative level of PlGF (e.g., as compared to the levelfrom a reference sample or standard or level) or to determine theabsolute concentration of PlGF as described above. Preferred assays forthe measurement of binding include fluorescence immunoassays.

In another example, an assay for detecting PlGF in a urine sampleincludes a membrane having a dehydrated labeled (e.g., for colorimetricdetection) PlGF binding agent (primary agent) and an immobilizedanti-PlGF binding agent (secondary agent). The membrane is exposed tothe sample. The sample rehydrates the labeled PlGF binding agent and ifPlGF is present in the sample, it will bind to the PlGF binding agent.The PlGF-primary agent complex will move down the membrane by capillarymovement and will interact with the immobilized secondary agent. Thisinteraction will produce a visible line from the colorimetric label atthe position at which the secondary agent is immobilized.

In another example, an assay for detecting PlGF in a urine sampleincludes a membrane having a dehydrated labeled (e.g., for calorimetricdetection) PlGF binding agent (primary agent), and an immobilizedanti-PlGF binding agent (secondary agent). The membrane also includespurified PlGF at a threshold concentration also immobilized on themembrane. In this example, the membrane is exposed to the urine sample.The sample rehydrates the labeled primary agent and if PlGF is presentin the sample at a concentration greater than the thresholdconcentration, it will bind to the PlGF binding agent. The PlGF-labeledprimary agent complex will move down the membrane by capillary movement.As the primary agent is already bound to the PlGF from the sample, itwill not bind to the immobilized purified PlGF and no visible line willappear at this “test” position. The PlGF-primary agent complex willcontinue down the membrane and will interact with the immobilizedsecondary agent. This interaction will produce a visible line from thecalorimetric label at the “control” position at which the anti-PlGFbinding agent is immobilized. In this example, only one visible linewill appear and will indicate a PlGF concentration above a thresholdconcentration. If the concentration of PlGF is below the thresholdconcentration, the labeled primary agent will bind to the immobilizedPlGF and a visible line will appear at this “test” location as well asat the “control” location. The test assay can also include multiple testlines aimed at detecting several concentrations of PlGF in the sample.Such a graded assay is described in U.S. Pat. No. 6,660,534.

In another example, a similar membrane based assay is used but is basedon standard sandwich ELISA methods. In this example, the membraneincludes a reaction zone having an immobilized primary PlGF bindingagent conjugated to an enzyme; a test zone having another immobilizedPlGF binding agent that binds to a region of PlGF not bound by the firstPlGF binding agent, and a control zone having an immobilized substancethat recognizes the primary PlGF binding agent. In both the test zoneand the control zone a detectable substrate for the enzyme conjugated tothe first immobilized PlGF binding agent is included. The membrane isexposed to the sample and the sample moves to the reaction zone bycapillary action. If PlGF is present in the sample, it binds to thefirst immobilized PlGF binding agent conjugated to an enzyme and forms acomplex which is then carried along by capillary flow to the test zone.The PlGF-immobilized PlGF binding agent conjugated to an enzyme complexthen binds to the second PlGF binding agent and forms a visible line atthe location of the immobilized second PlGF binding agent (the “test”zone). The remaining first PlGF binding agent is carried along bycapillary flow and will bind to the immobilized substance thatrecognizes or binds to the first binding agent and produce a visibleline at this location (the “control” zone). If PlGF is not present inthe sample, only the second line will appear at the control zone. Inpreferred embodiments, the first and second PlGF binding agents areantibodies and the agent that recognizes or binds to the first bindingagent is a secondary anti-immunoglobulin antibody that specificallyrecognizes the immunoglobulin of the first antibody. The intensity ofthe line in the test zone can be compared to assays using a standardamount of purified PlGF protein to determine if the sample contains PlGFabove or below a threshold concentration.

In any of the assays described herein, normal pregnant serum can be usedas an additional control and the activity of PlGF can be measured andquantified as a percentage of PlGF activity measured from normalpregnant serum.

For any of the assays described herein, the sample can be any bodilyfluid. A urine sample is preferred for the PlGF-based diagnostic assays.The membrane can be in a standard dipstick type format or lateral flowformat. The dipstick type of assay is known in the art for such assaysas pregnancy detection (measuring hormone levels in that case) orurinalysis detection of creatinine or albumin in the diagnosis of kidneydisease. Examples of various formats of dipstick type assays aredescribed in U.S. Pat. No. 6,660,534, incorporated herein by reference.

Any of the above PlGF detection assays can be used alone or incombination with additional diagnostic assays described herein or in theart. In a preferred embodiment, the PlGF diagnostic assay is used as aninitial screen followed by assays for the measurement of serum sFlt-1,PlGF and/or VEGF levels as described herein. In this way “at risk”patients can be identified and carefully monitored or screened furtherfor even greater diagnostic accuracy.

In preferred embodiments of any of the above-described PlGF-baseddiagnostic assays, the PlGF binding agent is preferably a primaryantibody that recognizes PlGF or a protein or peptide that interactswith PlGF. The secondary anti-PlGF binding agent is preferably asecondary antibody that recognizes the primary antibody or a proteinthat binds to the primary antibody (e.g., Protein A or Protein G), or anantibody that specifically binds the peptide that interacts with PlGF.In embodiments where the PlGF binding agent is labeled with an enzyme,the enzyme used preferably catalyzes a calorimetric reaction that can bedetected by eye and/or measured by spectrophotometry. Non-limitingexamples of preferred enzyme/substrate combinations are horseradishperoxidase/TMB, β-galactosidase/XGAL, and alkaline/phosphatase/1,2dioxetane. For embodiments that include a labeled PlGF binding agent,preferred labels include colorimetric (e.g., colloidal gold),chemiluminescent, or fluorescent labels.

In veterinary practice, assays may be carried out at any time during thepregnancy, but are, preferably, carried out early in pregnancy, prior tothe onset of pre-eclampsia symptoms. Given that the term of pregnanciesvaries widely between species, the timing of the assay will bedetermined by a veterinarian, but will generally correspond to thetiming of assays during a human pregnancy.

The diagnostic methods described herein can be used individually or incombination with any other diagnostic method described herein for a moreaccurate diagnosis of the presence of, severity of, or estimated time ofonset of pre-eclampsia or eclampsia. In addition, the diagnostic methodsdescribed herein can be used in combination with any other diagnosticmethods determined to be useful for the accurate diagnosis of thepresence of, severity of, or estimated time of onset of pre-eclampsia oreclampsia.

The diagnostic methods described herein can also be used to monitor andmanage pre-eclampsia or eclampsia in a subject. In one example, if asubject is determined to have a serum sFlt-1 protein level of 10 ng/mLand a serum level of free PlGF of 100 pg/mL, then VEGF can beadministered until the serum PlGF level rises to approximately 400pg/mL. In this embodiment, the levels of sFlt-1, PlGF, and VEGF, or anyand all of these, are measured repeatedly as a method of not onlydiagnosing disease but monitoring the treatment and management of thepre-eclampsia and eclampsia. As described above, in normal pregnancies,urinary levels of PlGF range from 200-800 pg/ml or 200-800 pg/mg ofcreatinine after 20 weeks of gestation. A value of PlGF less than 400pg/ml, preferably less than 200 pg/ml or 200 pg/mg of creatinine in aurine sample is considered diagnostic of preeclampsia or a propensity todevelop pre-eclampsia.

The invention also features diagnostic assays for the detection of acardiovascular condition or a propensity to develop a cardiovascularcondition. In a preferred embodiment, sFlt-1 levels are measured inwomen with a history of pre-eclampsia or eclampsia and compared tosFlt-1 levels from a reference sample. Reference samples preferablyinclude samples taken from women with previous pregnancies and nohistory of pre-eclampsia or eclampsia. Alterations in the levels ofsFlt-1 polypeptide or nucleic acid as compared to the reference samplecan be used to diagnose a cardiovascular condition or to predict apropensity to develop a cardiovascular condition. Alterations in thenucleic acid sequence of sFlt-1, PlGF, or VEGF as compared to areference sequence can also be used to diagnose a cardiovascularcondition or to predict a propensity to develop a cardiovascularcondition. Any of the diagnostic methods and metrics described above canbe used to monitor women with a history of pre-eclampsia or eclampsiapost-partum or to diagnose a cardiovascular condition or to predict apropensity to develop a cardiovascular condition. Post-partum monitoringcan be performed on a regular basis (e.g., once a month, once every sixmonths, yearly, every other year, or less frequently) to assist in thediagnosis, prediction, or prevention of future cardiovascular events orconditions.

Diagnostic Kits

The invention also provides for a diagnostic test kit. The diagnostictest kit includes the components required to carry out any of thediagnostic assays described above and instructions for the use of thecomponents to diagnose pre-eclampsia or eclampsia or the propensity todevelop pre-eclampsia or eclampsia. For example, a diagnostic test kitcan include antibodies to sFlt-1, VEGF, or PlGF, and components usefulfor detecting, and more preferably evaluating, binding between theantibodies and the sFlt-1, VEGF, or PlGF polypeptide. For detection,either the antibody or the sFlt-1, VEGF, or PlGF polypeptide is labeled,and either the antibody or the sFlt-1, VEGF, or PlGF polypeptide issubstrate-bound, such that the sFlt-1, VEGF, or PlGFpolypeptide-antibody interaction can be established by determining theamount of label attached to the substrate following binding between theantibody and the sFlt-1, VEGF, or PlGF polypeptide. In one example, thekit includes a PlGF binding agent and components for detecting thepresence of PlGF. A conventional ELISA or a sandwich ELSIA is a common,art-known method for detecting antibody-substrate interaction and can beprovided with the kit of the invention. sFlt-1, VEGF, or PlGFpolypeptides can be detected in virtually any bodily fluid including,but not limited to urine, serum, plasma, saliva, amniotic fluid, orcerebrospinal fluid. A kit that determines an alteration in the level ofsFlt-1, VEGF, or PlGF polypeptide relative to a reference, such as thelevel present in a normal control, is useful as a diagnostic kit in themethods of the invention. The kit can also include purified proteins tobe used as standards in the assay used to detect the level of sFlt-1,VEGF, or PlGF. Desirably, the kit will contain instructions for the useof the kit. In one example, the kit contains instructions for the use ofthe kit for the diagnosis of pre-eclampsia, eclampsia, or the propensityto develop pre-eclampsia or eclampsia. In another example, the kitcontains instructions for the diagnosis of cardiovascular conditions orthe propensity to develop cardiovascular conditions. In yet anotherexample, the kit contains instructions for the use of the kit to monitortherapeutic treatment or dosage regimens.

In one embodiment of the invention, such a kit includes a solid support(e.g., a membrane or a microtiter plate) coated with a primary agent(e.g., an antibody or protein that recognizes the antigen), standardsolutions of purified protein for preparation of a standard curve, abody fluid (e.g. serum or urine) control for quality testing of theanalytical run, a secondary agent (e.g., a second antibody reactive witha second epitope in the antigen to be detected or an antibody or proteinthat recognizes the primary antibody) conjugated to a label or an enzymesuch as horse radish peroxidase or otherwise labelled, a substratesolution, a stopping solution, a washing buffer and an instructionmanual.

Assays for Gene and Protein Expression

Blood serum from the subject is measured for levels of VEGF, PlGF, orany protein ligand known to bind to sFlt-1. Methods used to measureserum levels of proteins include ELISA, western blotting, orimmunoassays using specific antibodies. In addition, biological activitycan be determined using an in vitro angiogenesis assay to determine ifthe subject's blood has converted from an anti-angiogenic state to apro-angiogenic state. Such assays are described above in Example 2.

Blood serum samples from the subject can also be measured for levels ofVEGF, PlGF or sFlt-1 nucleic acid levels. There are several art-knownmethods to assay for gene expression. Some examples include thepreparation of RNA from the blood samples of the subject and the use ofthe RNA for northern blotting, PCR based amplification, or RNAseprotection assays.

Subject Monitoring

The disease state or treatment of a subject having pre-eclampsia,eclampsia, or a propensity to develop such a condition can be monitoredusing the methods and compositions of the invention. In one embodiment,the expression of an sFlt-1, VEGF, or PlGF polypeptide present in abodily fluid, such as urine, plasma, amniotic fluid, or CSF, ismonitored. Such monitoring may be useful, for example, in assessing theefficacy of a particular drug in a subject or in assessing diseaseprogression. Therapeutics that decrease the expression of an sFlt-1nucleic acid molecule or polypeptide or that increase the expression ofa VEGF or PlGF nucleic acid molecule or polypeptide are taken asparticularly useful in the invention.

Other Embodiments

From the foregoing description, it is apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference. In addition, U.S. application publicationnumber 2004-0126828 and PCT publication number WO2004/008946A2 arehereby incorporated by reference in their entirety.

1. A method of diagnosing a pregnant human subject as having, or havinga propensity to develop, pre-eclampsia or eclampsia, said methodcomprising: (a) measuring the level of free PlGF in a urine sample fromsaid pregnant human subject, wherein said free PlGF is a PlGFpolypeptide that has the ability to bind to sFlt-1; and (b) measuringthe level of sFlt-1 in a bodily fluid sample from said pregnant humansubject; wherein a level of free PlGF in said urine sample less than 400pg/ml and a level of sFlt-1 polypeptide greater than 2 ng/ml measuredduring mid-gestation or late gestation is a diagnostic indicator ofpre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.
 2. A method of diagnosing a pregnant human subject as having,or having a propensity to develop, pre-eclampsia or eclampsia, saidmethod comprising measuring the level of free PlGF in a urine samplefrom said pregnant subject, wherein said sample is obtained duringmid-gestation or late gestation, and measuring the level of creatininein said urine sample, wherein said free PlGF is a PlGF polypeptide thatthan 200 pg per mg of creatinine is a diagnostic indicator ofpre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.
 3. A method of diagnosing a pregnant human subject as havinga propensity to develop, early onset pre-eclampsia or eclampsia, saidmethod comprising measuring the level of free PlGF in a urine samplefrom said pregnant subject at 21-32 weeks and measuring the level ofcreatinine in said urine sample and comparing said level of free PlGFper mg of creatinine from said subject to the level of free PlGF per mgof creatinine from a reference sample, wherein said free PlGF is a PlGFpolypeptide that has the ability to bind to sFlt-1 and wherein adecrease of at least 50% in said free PlGF per mg of creatinine from thesubject sample compared to said reference sample is a diagnosticindicator of a propensity to develop early onset pre-eclampsia oreclampsia.
 4. The method of claim 3, wherein said reference sample is aprior sample taken from said subject.
 5. The method of claim 3, whereinsaid reference sample is a sample taken from a subject that is pregnantbut does not have pre-eclampsia or eclampsia, or a propensity to developpre-eclampsia or eclampsia.
 6. The method of claim 2, furthercomprising: (a) measuring the level of at least one of sFlt-1, freePlGF, and free VEGF polypeptide in a sample from said subject, whereinsaid VEGF is a VEGF polypeptide that has the ability to bind to sFlt-1and wherein said free PlGF is a PIGE polypeptide that has the ability tobind to sFlt-1 and wherein said sample is a bodily fluid selected fromthe group consisting of urine, blood, serum, and plasma; and (b)comparing said level of sFlt-1, free PlGF, or free VEGF from saidsubject sample to the level of sFlt-1, free PlGF, or free VEGFpolypeptide in a reference sample, wherein an increase of at least 10%in said level of sFlt-1 or a decrease of at least 10% in said level offree VEGF or free PlGF polypeptide from said subject sample compared tosaid reference sample is a diagnostic indicator of pre-eclampsia oreclampsia or a propensity to develop pre-eclampsia or eclampsia.
 7. Themethod of claim 6, wherein the level of sFlt-1 from a sample of serumfrom said subject is measured.
 8. The method of claim 6, wherein thelevel of sFlt-1 and free PlGF from a sample of serum from said subjectis measured.
 9. The method of claim 6, comprising measuring the level ofsFlt-1 and at least one of free VEGF and free PlGF and calculating therelationship between said levels of sFlt-1, and at least one of freeVEGF, and free PlGF using a metric, wherein an increase of at least 10%in the level of said sFlt-1 relative to at least one of said free VEGFand free PlGF level in said metric from said subject sample as comparedto said metric from a reference sample, diagnoses pre-eclampsia oreclampsia or a propensity to develop pre-eclampsia or eclampsia in saidsubject.
 10. The method of claim 9, wherein said metric comprises apre-eclampsia anti-angiogenic index (PAAI):[sFlt-1/VEGF+PlGF] and anincrease of at least 10% in said PAAI from the subject sample relativeto the reference sample diagnoses pre-eclampsia or eclampsia or apropensity to develop pre-eclampsia or eclampsia in said subject. 11.The method of claim 1, 2, or 3, wherein said measuring is done using animmunological assay.
 12. The method of claim 11, wherein saidimmunological assay is an ELISA.
 13. A method of diagnosing a pregnanthuman subject as having a propensity to develop early onsetpre-eclampsia or eclampsia, said method comprising (a) obtaining asample of urine from said subject; (b) contacting said sample with asolid support, wherein said solid support comprises an immobilized firstfree PlGF binding agent, for a time sufficient to allow binding of saidfirst free PlGF binding agent with free PlGF present in said sample,wherein said free PlGF is a PlGF polypeptide that has the ability tobind to sFlt-1; (c) contacting said solid support after step (b) with apreparation of a second labeled PlGF binding agent, for a timesufficient to allow binding of said second labeled PlGF binding agent tosaid free PlGF bound to said first immobilized free PlGF binding agent;(d) measuring the binding of said second labeled PlGF binding agent tothe immobilized free PlGF binding agent bound to free PlGF at theposition where the first free PlGF binding agent is immobilized; (e)measuring the level of creatinine in said subject sample; (f) comparingthe binding measured in step (d) with the binding measured using areference sample, and comparing the level of creatinine measured in step(e) with the level measured using a reference sample wherein a decreaseof at least 50% in the binding measured in step (d) for said sample, ascompared to the binding measured using a reference sample, whennormalized for said creatinine levels measured in step (e) is adiagnostic indicator of a propensity to develop early onsetpre-eclampsia or eclampsia.
 14. The method of claim 13, wherein saidreference sample is recombinant PlGF at a concentration of 400 to 800pg/ml.
 15. The method of claim 13, wherein said second labeled PlGFbinding agent comprises a colorimetric label or a fluorescent label. 16.The method of claim 13, wherein said free PlGF binding agent is anantibody, or purified fragment thereof, or a peptide.
 17. A method ofdiagnosing a pregnant human subject as having a propensity to developearly onset pre-eclampsia or eclampsia, said method comprising: (a)obtaining a sample of urine from said subject; (b) contacting saidsample with a solid support, wherein said solid support comprises animmobilized free PlGF binding agent that is detectably labeled, whereinsaid contacting is for a time sufficient to allow binding of said firstPlGF binding agent to free PlGF present in said sample, wherein saidfree PlGF is a PlGF polypeptide that has the ability to bind to sFlt-1;(c) measuring said labeled free PlGF binding agent bound to said freePlGF, wherein said measuring is capable of distinguishing between saidbound and unbound PlGF binding agent; (d) measuring the level ofcreatinine in said subject sample; and (e) comparing the bindingmeasured in step (c) with the binding measured using a reference sample,and comparing the level of creatinine measured in step (d) with thelevel measured using a reference sample, wherein a decrease of at least50% in the binding measured in step (c) for said sample, as compared tothe binding measured using a reference sample, when normalized for saidcreatinine levels measured in step (d) is a diagnostic indicator of apropensity to develop early onset pre-eclampsia or eclampsia.
 18. Themethod of claim 17, wherein the free PlGF binding agent comprises afluorescent label or a colorimetric label.
 19. The method of claim 13 or17, wherein said solid support is a membrane.
 20. The method of claim 3,13, or 17, further comprising measuring the level of sFlt-1, free PlGF,or free VEGF polypeptide, in a sample of bodily fluid from said subject,wherein said free VEGF is a VEGF polypeptide that has the ability tobind to sFlt-1 and wherein said free PlGF is a PlGF polypeptide that hasthe ability to bind to sFlt-1, and wherein said bodily fluid is selectedfrom the group consisting of urine, blood, serum, and plasma, wherein anincrease of at least 50% in the level of said sFlt-1 or a decrease of atleast 50% in the level of said free PlGF or free VEGF diagnoses saidsubject with a propensity to develop early onset pre-eclampsia oreclampsia.
 21. The method of claim 20, wherein said level of sFlt-1polypeptide is measured in a sample of serum from said subject.
 22. Themethod of claim 13 or 17, wherein said reference sample is a priorsample from said subject and a decrease of at least 50% in said levelsof free PlGF, when normalized for said creatinine levels, over time is adiagnostic indicator of a propensity to develop early onsetpre-eclampsia or eclampsia.
 23. The method of claim 1, 2, 3, or 6,further comprising determining the body mass index or gestational agefor said pregnant human subject.
 24. The method of claim 13, or 17,wherein said pregnant human subject is in the second trimester ofpregnancy.
 25. The method of claim 13, or 17, wherein said pregnanthuman subject is 21 to 32 weeks pregnant.
 26. The method of claim 2,further comprising: (a) measuring the levels of sFlt-1, free VEGF, andfree PlGF polypeptides in a sample from a human subject, wherein saidfree VEGF is a VEGF polypeptide that has the ability to bind to sFlt-1and wherein said free PlGF is a PlGF polypeptide that has the ability tobind to sFlt-1 and wherein said sample is a bodily fluid selected fromthe group consisting of urine, blood, serum, and plasma; and (b)calculating the relationship between said levels of sFlt-1, free VEGF,and free PlGF using a PAAI metric, wherein a PAAI value greater than orequal to 20 is a diagnostic indicator of pre-eclampsia or eclampsia. 27.The method of claim 6, wherein said reference sample is a prior sampletaken from said subject.
 28. The method of claim 27, wherein saidreference sample is taken during the first trimester of pregnancy. 29.The method of claim 6, wherein said reference sample is a sample takenfrom a subject that is pregnant but does not have pre-eclampsia oreclampsia, or a propensity to develop pre-eclampsia or eclampsia. 30.The method of claim 9, wherein said metric is sFlt-1/PlGF.
 31. Themethod of claim 22, wherein the prior sample is taken during the firsttrimester of pregnancy and the subject sample is taken during the secondor third trimester of pregnancy.
 32. The method of claim 13 or 17,wherein the reference sample is PlGF ranging from 10 pg/ml to 1 ng/mland a decrease of at least 50% in the binding measured in the subjectsample, when normalized to said creatinine levels, compared to thebinding measured using the reference sample is a diagnostic indicator ofa propensity to develop early onset pre-eclampsia or eclampsia.
 33. Themethod of claim 19, wherein said membrane is supported on a dipstickstructure and the sample is deposited on the membrane by placing thedipstick structure into the sample.
 34. The method of claim 4, whereinsaid prior sample is a sample taken during the first trimester ofpregnancy.
 35. The method of claim 6, 7, 8, or 21, wherein said sFlt-1is the level of free sFlt-1.
 36. The method of claim 6, wherein saidsFlt-1 is the level of bound sFlt-1.
 37. The method of claim 6, whereinsaid sFlt-1 is the level of total sFlt-1.
 38. The method of claim 10,wherein an increase of at least 90% in said PAAI from said subjectsample relative to the reference sample diagnoses pre-eclampsia oreclampsia or a propensity to develop pre-eclampsia or eclampsia in saidsubject.
 39. The method of claim 6, wherein an increase of at least 50%in the level of sFlt-1 or a decrease of at least 50% in the level offree VEGF or free PlGF polypeptide relative to said reference diagnosessaid subject as having a propensity to develop early onset pre-eclampsiaor eclampsia.
 40. The method of claim 9, wherein an increase of at least50% in the level of said sFlt-1 relative to at least one of said freeVEGF and free PlGF level in said metric from said subject sample ascompared to said metric from said reference sample diagnoses saidsubject as having a propensity to develop, pre-eclampsia or eclampsia.41. The method of claim 11, wherein said immunological assay comprisesan antibody, or purified fragment thereof, that specifically binds tothe sFlt-1 binding domain of PlGF.
 42. The method of claim 17, whereinsaid free PlGF binding agent is an antibody, or purified fragmentthereof, or a peptide.
 43. The method of claim 16 or 42, wherein saidantibody, or purified fragment thereof, specifically binds to the sFlt-1binding domain of PlGF.
 44. A method of diagnosing a pregnant humansubject as having a propensity to develop early onset pre-eclampsia oreclampsia, said method comprising measuring the level of free PlGF in aurine sample from said pregnant human subject, wherein the subject is inthe first or second trimester of pregnancy, and wherein said free PlGFis a PlGF polypeptide that has the ability to bind to sFlt-1, andcomparing said level of free PlGF from said subject to the level of freePlGF from a reference sample, wherein a decrease of at least 50% in saidfree PlGF from the subject sample compared to said reference sample is adiagnostic indicator of a propensity to develop early onsetpre-eclampsia or eclampsia.
 45. A method of diagnosing a pregnant humansubject as having or having a propensity to develop pre-eclampsia oreclampsia, said method comprising measuring the level of free PlGF in aurine sample from said pregnant human subject and measuring the level ofsFlt-1 in a bodily fluid sample from said pregnant human subject,wherein said free PlGF is a PlGF polypeptide that has the ability tobind to sFlt-1, and comparing said level of free PlGF and said sFlt-1from said subject to the level of free PlGF and said sFlt-1 from areference sample, wherein a decrease of at least 10% in said free PlGFand an increase of at least 10% in said sFlt-1 from the subject samplecompared to said reference sample is a diagnostic indicator of apre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.
 46. A method of diagnosing a pregnant human subject as havinga propensity to develop pre-eclampsia or eclampsia, said methodcomprising (a) obtaining a sample of urine from said subject, whereinsaid subject is less than 16 weeks pregnant; (b) contacting said samplewith a solid support, wherein said solid support comprises animmobilized first free PlGF binding agent, for a time sufficient toallow binding of said first free PlGF binding agent with free PlGFpresent in said sample, wherein said free PlGF is a PlGF polypeptidethat has the ability to bind to sFlt-1; (c) contacting said solidsupport after step (b) with a preparation of a second labeled PlGFbinding agent, for a time sufficient to allow binding of said secondlabeled PlGF binding agent to said free PlGF bound to said firstimmobilized free PlGF binding agent; (d) measuring the binding of saidsecond labeled PlGF binding agent to the immobilized free PlGF bindingagent bound to free PlGF at the position where the first free PlGFbinding agent is immobilized; and (e) comparing the binding measured instep (d) with the binding measured using a reference sample, wherein adecrease of at least 10% in the binding measured in step (d) for saidsample, as compared to the binding measured using a reference sample isa diagnostic indicator of pre-eclampsia or eclampsia or a propensity todevelop pre-eclampsia or eclampsia.
 47. A method of diagnosing apregnant human subject as having or having a propensity to developpre-eclampsia or eclampsia, said method comprising: (a) obtaining asample of urine from said subject, wherein said subject is less than 16weeks pregnant; (b) contacting said sample with a solid support, whereinsaid solid support comprises an immobilized free PlGF binding agent thatis detectably labeled, wherein said contacting is for a time sufficientto allow binding of said first PlGF binding agent to free PlGF presentin said sample, wherein said free PlGF is a PlGF polypeptide that hasthe ability to bind to sFlt-1; (c) measuring said labeled free PlGFbinding agent bound to said free PlGF, wherein said measuring is capableof distinguishing between said bound and unbound PlGF binding agent; and(d) comparing the binding measured in step (c) with the binding measuredusing a reference sample, wherein a decrease of at least 10% in thebinding measured in step (d) for said sample, as compared to the bindingmeasured using a reference sample is a diagnostic indicator ofpre-eclampsia or eclampsia or a propensity to develop pre-eclampsia oreclampsia.
 48. The method of claim 13 or 17, wherein the referencesample is PlGF ranging from 10 pg/ml to 1 ng/ml and wherein a bindingobserved in step (d) that is equal to or less than the binding observedfor 200 pg/ml of PlGF, when normalized for creatinine levels, is adiagnostic indicator of a propensity to develop early onsetpre-eclampsia or eclampsia.
 49. The method of claim 45, 46, or 47,wherein a decrease of at least 50% diagnoses said pregnant human subjectas having a propensity to develop early onset pre-eclampsia oreclampsia.
 50. The method of claim 1, 2, 6, 7, 24, 25, or 30, whereinthe method diagnoses said pregnant human subject as having a propensityto develop pre-eclampsia or eclampsia.
 51. The method of claim 1, 2, 6,7, 24, 25, or 30, wherein the method diagnoses said pregnant humansubject as having pre-eclampsia or eclampsia.
 52. The method of claim30, wherein the subject is 21-32 weeks pregnant and an increase of atleast 50% in the level of sFlt-1/PlGF diagnoses the subject as having apropensity to develop early onset pre-eclampsia or eclampsia.
 53. Themethod of claim 39 or 40, wherein said subject is 21-32 weeks pregnantand the method diagnoses the pregnant human subject as having apropensity to develop early onset pre-eclampsia.
 54. The method of claim6 or 26, wherein said VEGF polypeptide is selected from the groupconsisting of VEGF-A, VEGF-B, VEGF189, VEGF165, and VEGF121.
 55. Themethod of claim 1, 2, 3, 13, 17, or 44, wherein said PlGF polypeptide isan alternatively spliced isoform of PlGF.
 56. The method of claim 45,wherein said sFlt-1 is free or total sFlt-1.